OBJECTIVESThe primary objective was to evaluate the safety and local tolerance of a topical 2% (w/w) cidofovir gel, applied directly to the cervices of women with high-grade cervical intraepithelial neoplasia (CIN 2+). The secondary objective was to evaluate the pharmacokinetics of cidofovir during the treatment.MATERIALS AND METHODSNine women with CIN 2+, were treated with a course of 3 g of cidofovir gel, applied locally once per week for 3 weeks in total (9 g). The treatment was administered in a cervical cap, applied to the cervix for 5 or 10 hours (n = 6 and 3 patients, respectively). Follow-up included a structured questionnaire, a gynecological examination, blood analysis for hematology, C-reactive protein (CRP), and renal function assessment plus pharmacokinetic analyses of cidofovir after each treatment and at the end of the full course.RESULTSNo clinically significant hematological/biochemical abnormalities or serious adverse events (SAE) were reported, although 6 mild to moderate adverse events (AE) occurred in relation to the study drug: 1 flu-like syndrome and 5 local AEs. Plasma concentrations of cidofovir were very low (mean Cmax of 103.0 and 99.2 ng/mL after 5 and 10 hours of exposure, respectively).CONCLUSIONCidofovir, directly applied on CIN 2+, is reasonably well tolerated and the systemic exposure following topical application is much lower than that seen with intravenous administration, at the approved dose.
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Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and fetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at 10.5-11.5 days postcoitum (dpc). Knowing the key contribution of PAI-1 in the regulation of angiogenesis, we have now analyzed the consequence of PAI-1 deficiency on murine placentation. Morphological and quantitative computer-assisted image analysis revealed abnormal placental morphology in PAI-1-/- mice at 10.5 and 12.5 dpc. At 10.5 dpc, the genetic ablation of PAI-1 resulted in a transient reduction of both maternal and fetal vascularizations in the placenta and increased trophoblast cell density. This was associated with a poorer development of the labyrinth and an extension of the decidua. A larger spongiotrophoblast layer appeared at 12.5 dpc in PAI-1-deficient mice. Placental morphology was normalized at 14.5 dpc. Microarray analyses performed on laser capture microdissected labyrinths revealed that 46 genes were differentially expressed between the two genotypes at 10.5 dpc. However, only 11 genes were still differently modulated at 14.5 dpc, when normalization of placental morphology had taken place. This transcriptomic profiling highlighted a dysregulation in the expression of placenta-related cathepsin family members. Altogether our data provide evidence for a transient impaired placental morphology in PAI-1-deficient mice that is then normalized, leading to normal embryonic development.

Matrix metalloproteinases (MMPs) are principal participants in tumor development. In addition to serve as a useful biochemical marker, MMP expression may also provide a target for the diagnostic in vivo imaging of tumors, using a radiolabeled inhibitor. This study investigates the use of membrane type 1 (MT1)-MMP as target for in vivo tumor diagnosis. Specific binding of the endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2) to MT1-MMP has been previously described. In this study, biodistribution and imaging experiments were performed on MT1-MMP-overexpressing (S.1.5) and control (C.IV.3) tumor-inoculated mice using [(123)I]-recombinant human TIMP-2 (rhTIMP-2) as radioligand and [(123)I]-rhTIMP-1 as control. The expression profile was controlled in vitro and on tumor extracts. rhTIMP-2 as well as rhTIMP-1 were labeled using the Iodogen method and characterized. Biodistribution of [(123)I]-rhTIMP-2 showed a tumor uptake of 2.87% ± 1.58% ID/g at 3 hours postinjection in S.1.5. Tumor values of [(123)I]-rhTIMP-1 and [(123)I]-rhTIMP-2 evaluated in S.1.5 and C.IV.3, respectively, were significantly lower. Planar imaging revealed significant uptake of [(123)I]-rhTIMP-2 in S.1.5 compared with contralateral background areas. This could not be observed in C.IV.3 and with [(123)I]-rhTIMP-1 in S.1.5. All tumors were well established (200-800  mg). These results suggest that rhTIMP-2 holds potential for development of radiotracers for in vivo imaging in overexpressing MT1-MMP but not in similar tumors that do not express this protease.

G-CSF in individual follicular fluids correlates with the potential of the corresponding embryo to result in a live birth after transfer in IVF. To evaluate the requirements for routine follicular fluid G-CSF quantification, we compared follicular fluid G-CSF measurements made with two multiplexed microbead assays purchased from Bio-Rad Laboratories and R&D Systems, and a commercial G-CSF ELISA (R&D Systems). Individual follicular fluids (n=139) associated with transferred embryos were analysed to determine cytokine profile and the fate of each transferred embryo was recorded. The effect of multiplexing as well as comparison of the respective performances of the microbead assay with a flow cytometry assay was explored. Multivariable logistic regression analysis was performed and receiver operating characteristic (ROC) analysis was used to determine the performance and sensitivity/specificity of each method for individual follicular fluids. Covariate factors known to influence IVF outcome such as age, serum oestradiol and embryo score were systematically integrated in each analysis. The quantification of follicular fluid G-CSF using microbead assay methodologies, but not ELISA, yielded results showing the utility of follicular fluid G-CSF as a biomarker predictive of a successful delivery (Auroc: 0.77 [0.68–0.84] (p=0.003) and 0.75 [0.66–0.82] (p=0.004) for Bio-Rad and R&D Systems microbead assays respectively), whereas follicular fluid G-CSF values quantified by ELISA were not predictive (Auroc:0.61 [0.52–0.70] p=0.84). Microbead assay and flow cytometry appeared similarly efficient for quantifying follicular fluid G-CSF and multiplex versus single-plex assays did not influence the reliability of quantification.

The development of blood vessels inside tumors is required to provide the nutrients and oxygen needed for tumor growth and to allow the spread of cancer cells at a distance to form metastasis. Angiogenesis is also implicated in ocular diseases like age‐related macular degeneration. The present work describes the potential anti‐angiogenic properties of a coumarinic derivative, 3‐bromophenyl 6‐acetoxymethyl‐2‐oxo‐2H‐1‐benzopyran‐3‐carboxylate (IK9), previously described as a potent inhibitor of HT 1080 fibrosarcoma cell invasion in vitro and tumor growth in vivo. In vivo, ex vivo, and in vitro models were used to delineate the anti‐angiogenic properties of IK9. The anti‐angiogenic effect of IK9 was demonstrated in vivo in a choroidal neovascularization mice model and additionally ex vivo in a rat aortic ring assay where it was more active than the known matrix metalloproteinase inhibitor Ro 28‐2653. IK9 did not affect apoptosis, proliferation, or endothelial cell invasiveness in vitro. These findings suggest a complex mechanism of action of the compound via direct or indirect effects on endothelial cell properties. This study identifies IK9 as a new potent inhibitor of angiogenesis and suggests its potential use as a therapeutic agent. Drug Dev Res 71, 2010. © 2010 Wiley‐Liss, Inc.

Unraveling the molecular mechanisms of lymphangiogenesis is hampered by the lack of appropriate in vitro models of three-dimensional (3D) lymph vessel growth which can be used to exploit the potential of available transgenic mice. We developed a potent reproducible and quantifiable 3D-culture system of lymphatic endothelial cells, the lymphatic ring assay, bridging the gap between 2D-in vitro and in vivo models of lymphangiogenesis. Mice thoracic duct fragments are embedded in a collagen gel leading to the formation of lymphatic capillaries containing a lumen as assessed by electron microscopy and immunostaining. This assay phenocopies the different steps of lymphangiogenesis, including the spreading from a preexisting vessel, cell proliferation, migration and differentiation into capillaries. Our study provides evidence for the implication of an individual matrix metalloproteinase, MMP-2, during lymphangiogenesis. The lymphatic ring assay is a robust, quantifiable and reproducible system which offers new opportunities for rapid identification of unknown regulators of lymphangiogenesis.

Novel 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives were synthesized and their potency in reducing the invasive behaviour of HT 1080 fibrosarcoma cells was evaluated. Structure-activity relationships were deduced from biological results and will be used in further design of new active compounds. In particular, the acetoxymethyl substituent found at the 6-position of previously described active compounds can be replaced by an acetamidomethyl substituent without loss of potency; while the presence of an aryl ester function at the 3-position was preferred to a thioester or an amide function to induce marked biological activity. This work confirms the interest of aryl esters of 6-substituted coumarin-3-carboxylic acids as potential new anti-cancer agents.

Aim: Decidual endothelial cells are able to synthesize C1q and express surface‐bound C1q under physiological conditions. Since decidua is a site of active angiogenesis, we sought to ascertain whether C1q could play a role in this process.Material and Methods: To confirm our hypothesis we used different approaches such as permeability, cell migration and proliferation assay, besides wound healing and aortic ring assay.Results: C1q acts as a permeabilizing factor inducing the FITC‐BSA leakage through a monolayer of endothelial cells (ECs). Next, we found that C1q was able to promote motility of ECs in a wound healing assay, and to recruit ECs acting as a chemotactic factor, furthermore C1q was also found to have an additional effect on EC inducing cell proliferation. To confirm and extend these data, we used the rat aortic ring assay to evaluate the ex vivo effect of C1q.Conclusion: Based on our findings, we propose that C1q exerts unexpected proangiogenic effects.

A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay—a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.

BACKGROUND:The levonorgestrel-releasing intrauterine system (LNG-IUS) is an effective contraceptive and has many non-contraceptive health benefits. However, it is commonly associated with irregular endometrial bleeding. Metalloproteinases contribute to extracellular matrix (ECM) remodelling and regulate bleeding during the menstrual cycle. Enhanced metalloproteinase expression participates in the pathogenesis of breakthrough bleeding. Thus the objective of this study was to compare matrix metalloproteinase (MMP) expression in endometrium during luteal phase and in short-term (1 month) and long-term (> or =6 months) LNG-IUS users.METHODS:MMP expression was analysed by semi-quantitative RT-PCR and immunohistochemistry. Gelatinase activity was determined by gelatin zymography.RESULTS:MMP-1, -2, -3, -7, -9 and -12 mRNAs levels were increased, whereas that of MMP-26 was decreased in the endometrium of LNG-IUS users. MMP-1, -2, -3, -7 and -9 were localized by immunohistochemistry in all biopsies in the short-term group but in only 0-27% in the control group. The incidence of positive immunostaining for MMP-2 and -3 decreased significantly in the long-term compared with short-term LNG-IUS users. MMP-26 was localized in all biopsies from the control group but in only 14 and 25% from the short- and long-term LNG-IUS groups, respectively. In both LNG groups, the numbers of macrophages (the major source of MMP-12) was increased.CONCLUSIONS:MMP-1, active MMP-2, MMP-3, MMP-7, MMP-9 and MMP-12 are more prevalent in the short-term LNG-IUS group, suggesting their important contribution to ECM breakdown and transient bleeding. The decrease in the percentage of women expressing MMP-2 and -3 might contribute to the decreased occurrence of unwanted spotting and bleeding in long-term LNG-IUS users.