Does Plasminogen Activator Inhibitor-1 Drive Lymphangiogenesis?

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis

The lymphatic ring assay: a 3D-culture model of lymphangiogenesis.

peer reviewedLymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies1 and understanding the molecular and cellular basis of this complex process is essential for the development of novel therapeutic strategies. Studies on lymphangiogenesis have been hampered by difficulties in culturing lymphatic capillaries as three-dimensional (3D) structures in vitro that mimic the in vivo features of lymphatic vessels and lymphangiogenesis. The lymphatic ring assay described here phenocopies the different steps of lymphangiogenesis, including the spreading from a preexisting vessel, cell proliferation, migration and differentiation into capillaries. It consists on the adaptation of the aortic ring assay that has proved to be useful to investigate the molecular basis of angiogenesis2-4. The lymphatic ring model is an ideal assay for testing the activity of lymphangiogenic agonists or antagonists. The absence of inflammatory cells allows a simple interpretation of results and the determination of direct effects of compounds on lymphatic endothelial cell properties. Another advantage of the lymphatic ring assay is that cell outgrowing are primary cells which have not been modified by repeated passages or immortalization. This culture model bridges the gap between in vitro and in vivo studies and allows genetic analysis by using thoracic ducts from genetically modified mice

La régulation de l'expression des gènes du virus de la varicelle et du zona

Varicella-zoster virus (VZV) belongs to the alphaherpesvirus family and shares many important structural and functional similarities with other members of the family such as herpes simplex virus type 1 (HSV-1). VZV is responsible for two different clinical syndromes, varicella which is the result of the primary infection and zoster which is due to virus reactivation remaining latent in the peripheral nervous system. VZV DNA is 124,884 base pair long and encodes four regulatory proteins (IE4, IE61, IE62 and IE63). Using transient expression systems, we have shown that IE4, IE62 and IE63 can regulate the expression of an indicator gene driven by various VZV promoter regions, demonstrating that these proteins play important roles in the infectious cycle

Immuno PCR quantitative pour la détection et la quantification de la protéine prion

Immuno-polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno-quantitative PCR (iqPCR), exploiting real-time PCR technology, in order to improve this immuno-detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post-mortem stage

Lymphoid cell apoptosis induced by trophoblastic cells: a model of active foeto-placental tolerance

To test the hypothesis that CD95-L (Fas-L) present on trophoblastic cells plays a part in establishing foeto-placental tolerance by inducing apoptosis of immune defence cells, we cocultured trophoblasts with lymphoid cells and scored the frequency of cell death in these cultures. We prepared human trophoblastic cells from term placentas removed by C-section and placed them in culture for 48 h before introducing the lymphoid cells. We added Jurkat cells, a CD3 + lymphoid cell line, or purified T cells from human blood to the cultured trophoblasts and monitored apoptosis by electron microscopy and flow cytometry after TUNEL or annexin V labelling. The frequency of cell death in the CD3 + cell population was higher when the lymphoid cells were cocultured with trophoblastic cells than when they were cultured alone. This frequency increased with time but was reduced when anti-CD95-L antibodies were added to the culture medium. Cell death was less frequent in the lymphoid cell population when trophoblasts were replaced with human fibroblasts not expressing CD95-L. (C) 1999 Elsevier Science B.V. All rights reserved

Modeling lymphangiogenesis in a three-dimensional culture system

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.status: publishe

Tumor development after orthotopic injection of VEGF-C overexpressing MCF7 cells or control MCF7 cells implanted in the mammary fat pads (mfp) of PAI-1 WT or PAI-1^−/− mice.

<p>(A): RT-PCR analysis of VEGF-C and 28S mRNA expression by MCF7. (B): Tumor incidence (%) is defined as the percentage of palpable tumor per mfp. (C): Tumor volume was measured as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009653#s2" target="_blank">Methods</a>. (D): Representative figure of a typical metastasis in lymph node (left) and lung (right). (E): Percentage of animal bearing at least a tumor nodule (metastasis% detected in lymph nodes (black boxes) and lungs (white boxes). Number of mfp per condition = 34–38. The mice PAI-1 status (WT or −/−) and the VEGF-C production (VEGF-C) or not (Ctl) by MCF7 cells are indicated below each graph. Data are ± S.E.M. Scale bars: 200 µm. ** P≤0.01, *** P≤0.001, NS = Non Significant.</p

Development of lymphangioma in PAI-1 WT or PAI-1^−/− mice.

<p>A macroscopic decrease of lymphangioma is observed in PAI-1^−/− as compared to WT (A). Similar recruitment of lymphatic vessels (assessed by Lyve-1 positivity) (B), and of CD45 positive-inflammatory cells (C) was observed in both genotypes. Evaluation of fibrosis was performed by Sirius red staining (D). Representative images are shown on the left and quantifications performed by computerized image analysis are shown on the right Data are ± S.E.M (n = 6). Scale bars: A = 0.5 mm, B–D = 200 µm, ** P≤0.01.</p