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Matrix metalloproteinases (MMPs) constitute a large family of extracellular matrix degrading proteases implicated in a number of physiological and pathological processes, including angiogenesis. However, the relative importance of the individual MMPs in vessel formation is poorly understood. Using the three-dimensional rat aortic model, the role of the MMPs in angiogenesis in vitro was investigated both by the use of synthetic MMP inhibitors, and by a study of the expression of nine MMPs and three of their endogenous inhibitors (the TIMPs) during vessel formation. Inhibition of microvessel growth in this model by the MMP inhibitor Marimastat demonstrated the requirement of the MMPs for angiogenesis in both collagen and fibrin matrices (half-maximal inhibition at 5 and 80 nM, respectively). The profile of MMP expression was seen to be modified by both matrix composition and exogenous growth factors. For example, whilst the gelatinase MMP-2 and stromelysin MMP-3 were present at high levels in fibrin culture, the stromelysin MMP-11 and membrane-type-1-MMP were more highly expressed during vessel formation in collagen. The angiogenic basic fibroblast growth factor (bFGF) upregulated the expression of the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) and the interstitial collagenase MMP-13, whereas vascular endothelial growth factor (VEGF) led to a marked increase in expression of MMP-2 only. Together, the environment-dependent upregulation in expression of a number of MMPs during angiogenesis, and the total inhibition of vessel growth observed at nanomolar concentrations of synthetic MMP inhibitors, suggests a major collective role of these enzymes in angiogenesis, and provides a basis for further development of MMP inhibitors for anti-angiogenic therapy.

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The study of the angiogenic process and the search for novel therapeutic agents to inhibit, or stimulate, angiogenesis has employed a wide range of in vivo 'angiogenesis' assays (reviewed in 1-3). These differ greatly in their difficulty, quantitative nature, rapidity, and cost. The classical in vivo models include the rabbit ear chamber, hamster cheek pouch, dorsal skin chamber, dorsal skin and air-sac model, anterior chamber/iris and avascular corneal pocket assay, and the chick embryo chorioallantoic membrane (CAM) assay. More recent methods involve the implantation of preloaded Matrigel or alginate plugs, or collagen or poly vinyl sponges (1). Largely owing to its simplicity and low cost, the CAM is the most widely used in vivo model for the study of both angiogenesis and antiangiogenesis (1,4).

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Angiogenesis is a necessary component of normal tissue repair, tumor growth and dissemination, and a wide variety of other inflammatory and pathological processes as well, including diabetic retinopathy, rheumatoid arthritis, and psoriasis. Consequently, the last two decades have seen extensive research into the regulation of neovascularization, particularly in tumors. Partly due to the emphasis on tumor angiogenesis, much of this effort has been devoted to the detection and characterization of angiogenic growth factors and inhibitory molecules. Thus assays have commonly been developed to measure the amount of vascular growth in vivo, or the modulation of endothelial cell (EC) proliferation, or migration in simple 2D culture systems (1).

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In vitro angiogenesis assays are essential for the identification of potential angiogenic agents and screening for pharmacological inhibitors. Among these assays, the rat aortic ring model developed by Nicosia bridges the gap between in vivo and in vitro models. The quantification of angiogenesis on this system must be applicable to characterise vascular networks of various states of complexity. We present here an improved computer-assisted image analysis which allows: (1) the determination of the aortic ring area and its factor shape; (2) the number of microvessels, the total number of branchings, the maximal microvessel length and the microvessel distribution; (3) the total number of isolated fibroblast-like cells and their distribution. We show that this method is suitable to quantify spontaneous angiogenesis as well as to analyse a complex microvascular network induced by various concentrations of vascular endothelial growth factor (VEGF). In addition, by evaluating a new parameter, the fibroblast-like cell distribution, our results show that: (1) during spontaneous angiogenic response, maximal fibroblast-like cell migration delimits microvascular outgrowth; and (2) the known angiogenic inhibitor Batimastat prevents endothelial cell sprouting without completely blocking fibroblast-like cell migration. Finally, this new method of quantification is of great interest to better understand angiogenesis and to test pro- or anti-angiogenic agents.

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In an attempt to improve the relevance of human tumour xenografts to the clinical situation, we have established 3 models of human ovarian carcinoma (IGROV1, A2780 and NIH:OVCAR-3) in nude mice in which progressive peritoneal carcinomatosis resulted in the death of tumour-bearing animals (median survival times: 32, 40 and 64 days, respectively). Histological analyses revealed both common and different characteristics in growth patterns and dissemination profiles. In each case, three stages of the disease were defined (early, intermediate and late). The antitumour activities of adriamycin, cisplatin, cyclophosphamide and paclitaxel were then compared when administered at the early stage where small multifocal tumour nodules were detectable in the peritoneal cavity of the animals. Significant antitumour activities of cisplatin and particularly paclitaxel were noted in terms of increase in survival time of the treated mice (T/C values for IGROV1, A2780 and NIH:OVCAR-3 respectively: 152%, 167%, and 187% for cisplatin and 211%, 179% and >283% for paclitaxel), paclitaxel being curative against the NIH:OVCAR-3 xenograft. These results reflect the high efficacy of these two drugs in the clinic in the treatment of ovarian carcinoma. The clinically used CA125 tumour marker, not detectable in healthy mice, was measured in the serum of mice bearing IGROV1 and NIH:OVCAR-3 tumours. CA125 serum levels increased as a function of time and were well correlated to disease progression. Moreover, treatment with cisplatin and paclitaxel led to significant decreases in these levels of between 58% and 100%. This human serum marker could be used to predict early on the efficacy of chemotherapy in these two models. In conclusion, the three experimental ovarian carcinomas possess several important characteristics of the human disease and may thus be used as a screen to select new antitumour drugs potentially active in this pathology.

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The microenvironment of the majority of solid tumours in which new vessels must grow and survive is acidic. Whilst recent reports suggest a role of the low tumour pH in the invasive and metastatic potential of tumour cells, little is known as to its impact on angiogenesis. The three-dimensional in vitro rat aortic ring model was used to study the effects of low extracellular pH (pH(e)) on microvascular growth. The spontaneous angiogenic response in collagen gels was seen to be highly dependent on the pH of the culture medium, with optimal outgrowth at pH 7.4, and a marked delay in microvascular growth at pH 6.9. This inhibition of vascular development was reversible. The absence of similar effects of medium pH on monolayer outgrowths of endothelial cells from rat aortic rings suggested an effect of pH(e) on aspects specific to three-dimensional growth. Vascular endothelial growth factor and basic fibroblast growth factor, whilst having limited effects at pH 7.4, were seen to reduce the time to onset of vessel outgrowth at pH 7.1, and lead to an initial growth rate similar to that observed at pH 7.4 in the absence of growth factors. Thus, the low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pH(e) when stimulated by exogenous growth factors, high concentrations of which would be present in vivo.

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The antitumour activity of S 16020-2, a new topoisomerase II inhibitor, was evaluated in comparison with doxorubicin against 13 human tumours, including colon (HT-29, Colo320DM), breast (MCF7, MDAMB-231), ovary (SK-OV-3, A2780, NIH:OVCAR-3), non-small cell lung (NCI-H460, A549, Calu-6, NCI-H125) and small-cell lung (NCI-H69, SCLC6) cancers. S 16020-2 was administered weekly intravenous within a dose range of 20-90 mg/kg for 3 weeks. Antitumour responses were obtained in all the tumour types tested except in the two colon cancers. S 16020-2 produced significant growth delays in nine tumour models and induced regressions of all A549 lung tumours. The antitumour activity of S 16020-2 was superior to that of doxorubicin against the NCI-H460, A549, NCI-H69, SCLC6 and NIH:OVCAR-3 xenografts. These results demonstrate the broad spectrum of antitumour activity of S 16020-2 in a large panel of in vivo experimental models and confirm its interest as a potential agent in the treatment of malignant disease.

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