Francis Frankenne

MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression

Membrane type 1 metalloprotease (MT1‐MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro‐MMP 2. We investigated the effects of MT1‐MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1‐MMP or MMP‐2. MT1‐MMP and MMP‐2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1‐MMP 1) were able to activate endogenous or exogenous pro‐MMP‐2, 2) displayed an enhanced in vitro invasiveness through matrigel‐coated filters independent of MMP‐2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutanously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1‐MMP regardless of MMP‐2 expression levels, suggesting that the production of MMP‐2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1‐MMP‐producing cells was associated with an up‐regulation of VEGF expression. These results emphasize the importance of MT1‐MMP during tumor angiogenesis and open new opportunities for the development of anti‐angiogenic strategies combining inhibitors of MT1‐MMP and VEGF antagonists.—Sounni, N. E., Devy, L., Hajitou, A., Frankenne, F., Munaut, C., Gilles, C., Deroanne, C., Thompson, E. W., Foidart, J. M., Noel, A. MT1‐MMP expression promotes tumor growth and angiogenesis through an up‐regulation of vascular en‐dothelial growth factor expression. FASEB J. 16, 555–564 (2002)

MMP-2- and MMP-9-Linked Gelatinolytic Activity in the Sputum from Patients with Asthma and Chronic Obstructive Pulmonary Disease

Background: The course of asthma and chronic obstructive pulmonary disease (COPD) is associated with bronchial morphological changes. Metalloproteinases are thought to play a role in these structural changes. Methods: We studied the gelatinolytic activity present in the induced sputum from 20 patients with asthma, 20 with COPD and 19 healthy controls. The assessment of gelatinolytic activity was performed by quantitative zymography, and gelatinolytic species were identified by Western blot analysis. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was detected by reverse zymography and ELISA. Results: From zymography, we found significantly higher gelatinolytic activity linked to pro-matrix metalloproteinase-9 (pro-MMP-9) in the sputum from asthmatics (p < 0.0001) and COPD patients (p < 0.0001) compared to the control group. Furthermore, the activated form of MMP-9 (85 kD) was found in the sputum from 60% of asthmatics and 85% of COPD patients, but was absent in that of control subjects (p < 0.0001). Importantly, although less frequently detectable than pro-MMP-9, pro- MMP-2 (72 kD) was found more frequently in asthmatics (50%) than in control subjects (5%) (p < 0.005). We also described two unusual gelatinolytic species of 45 and 120 kD and showed that they derived from MMP-9 according to their ability to bind gelatin and anti-MMP-9 antibody. Levels of TIMP-1 were higher in asthmatics (p < 0.05) and COPD patients (p < 0.05) than in controls. Conclusion: Asthmatics and COPD patients display an increased gelatinolytic activity linked to MMP-2 and MMP-9 and higher levels of TIMP-1 in their sputum.

Placental Growth Hormone Levels in Normal Pregnancy and in Pregnancies with Intrauterine Growth Retardation

ABSTRACT: To assess the possible role of placental growth hormone (GH) in fetoplacental growth, we measured placental and pituitary GH (GHN) in maternal plasma by means of two RIA using two MAb (5B4 recognizing both placental GH and GHN, and K24 recognizing only GHN) during pregnancy. IGF-I also was measured by RIA in the same samples after extraction. A transverse study of 186 samples obtained between 8 wk of amenorrhea (WA) and term confirmed the reported rise in GH immunoreactivity with 5B4 after 24 to 25 WA from 12.3 ± 2.0 mU/L (mean ± SEM) to a plateau of 27.5 ± 3.4 mU/L at 34 to 35 WA together with the decrease in GHN to undetectable levels by 24 to 25 WA. IGF-I levels increased from 164.0 ± 44.6 μUg/L at 24 to 25 WA to 331.6 ± 63.6 μUg/L at term. A longitudinal study of 31 normal pregnant women confirmed this hormonal pattern and the reported placental GH plateau after 35 WA. A drastic decrease in placental GH was observed with the onset of labor (from 26.9 ± 2.1 to 2.7 ± 1.1 mU/L), whereas the decrease in IGF-I was not significant (from 212.9 ± 26.5 to 162.4 ± 16.9 μUg/L). Interestingly, maternal plasma samples obtained after 31 WA until the initiation of labor in 22 cases of intrauterine growth retardation (six cases of toxemia, one chromosomal aberration, one maternofetal infection, 14 idiopathic) contained significantly lower amounts of placental GH (14.9 ± 1.6 mU/L versus 26.5 ± 1.2 mU/L in normal pregnancies; p < 0.001). Plasma IGF-I levels were also lower than normal (156.0 ± 25.5 μUg/L versus 285.1 ± 40.8 μUg/L; p < 0.001). These results suggest a relationship between placental GH levels in the maternal plasma and the development of the fetoplacental unit.

Growth hormone 24‐h serum profiles during pregnancy—lack of pulsatility for the secretion of the placental variant

Summary. Serum profiles of growth hormone (GW) were recorded for 24 h in women at different stages of normal pregnancy. Two monoclonal antibodies directcd against different epitopes and unaffected by human placental lactogen were used in radioimmunoassays to distinguish the pituitary 22K‐GH from the placental GH variant. The ‘normal’ episodic peak activity of GH in non‐pregnant and first trimester pregnant women was dramatically changed into a continuous very stable secretion during late pregnancy. This change was first observed at 17 weeks gestation. It is concluded that during the second half of pregnancy, serum measurements of GH reflect a major contribution from a non‐cpisodically secreted placental GH variant and a concomitant suppression of pituitary GH. This specific signal, i.e. a continuous GH secretion, may be an important regulator of maternal liver metabolism during pregnancy.

Expression Pattern of Metalloproteinases and Tissue Inhibitors of Matrix-Metalloproteinases in Cycling Human Endometrium

Abstract The cyclic growth, differentiation, and cell death of endometrium represents the most dynamic example of steroid-driven tissue turnover in human adults. Key effectors in these processes—matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs)—are regulated by ovarian steroids and, locally, by cytokines. We used reverse transcription-polymerase chain reaction to evaluate the expression of both transcriptionally regulated molecules such as estrogen receptor-α, progesterone receptor, and prolactin and a large array of MMPs and TIMPs (MMP-1, -2, -3, -7, -8, -9, -11, -12, -19, -26, MT1-MMP, MT2-MMP, MT3-MMP, TIMP-1, -2, -3). Altogether, three distinct patterns of MMP and two patterns of TIMP expression were detected in cycling endometrium: 1) MMPs restricted to the menstrual period (MMPs-1, -3, -8, -9, -12); 2) MMPs and TIMPs expressed throughout the cycle (MMP-2, MT1-MMP, MT2-MMP, MMP-19, TIMP-1, and TIMP-2); 3) MMPs predominantly expressed during the proliferative phase (MMP-7, MMP-11, MMP-26, and MT3-MMP); and 4) TIMP-3, which, contrary to the other TIMPs, shows significant modulations, with maximum expression during the late secretory and menstrual phases. These specific patterns of MMP expression associated with each phase of the cycle may point to specific roles in the processes of menstruation, housekeeping activities, angiogenesis, tissue growth, and extracellular matrix remodeling.

Anti-Invasive, Antitumoral, and Antiangiogenic Efficacy of a Pyrimidine-2,4,6-trione Derivative, an Orally Active and Selective Matrix Metalloproteinases Inhibitor

Purpose: The implication of matrix metalloproteinases (MMPs) in the major stages of cancer progression has fueled interest in the design of synthetic MMP inhibitors (MMPIs) as a novel anticancer therapy. Thus far, drugs used in clinical trials are broad-spectrum MMPIs the therapeutic index of which proved disappointingly low. The development of selective MMPIs for tumor progression-associated MMPs is, thus, likely to offer improved therapeutic possibilities. Experimental Design: The anti-invasive capacity of a series of pyrimidine-trione derivatives was tested in vitro in a chemoinvasion assay, and the most potent compound was further evaluated in vivo in different human tumor xenograft models. The activity of this novel selective MMPI was compared with BB-94, a broad-spectrum inhibitor. Results: Ro-28-2653, an inhibitor with high selectivity for MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, showed the highest anti-invasive activity in vitro. In vivo, Ro-28-2653 reduced the growth of tumors induced by the inoculation of different cell lines producing MMPs and inhibited the tumor-promoting effect of fibroblasts on breast adenocarcinoma cells. Furthermore, Ro-28-2653 reduced tumor vascularization and blocked angiogenesis in a rat aortic ring assay. In contrast, BB-94 up-regulated MMP-9 expression in tumor cells and promoted angiogenesis in the aortic ring assay. Conclusion: Ro-28-2653, a selective and orally bioavailable MMPI with inhibitory activity against MMPs expressed by tumor and/or stromal cells, is a potent antitumor and antiangiogenic agent. In contrast to broad-spectrum inhibitors, the administration of Ro-28-2653 was not associated with the occurrence of adverse side effects that might hamper the therapeutic potential of these drugs.

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

BACKGROUND 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 beta1 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% O(2)) or hypoxia (1% O(2)). 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% O(2) (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.

Presence of Oestrogen Receptor Type Beta in Human Retina

BACKGROUND/AIMS Recent studies have demonstrated the existence of two oestrogen receptor subtypes α (ORα) and β (ORβ) with significant differences of expression among organs. Since important pathologies of human eye could be linked to hormonal status, the expression of ORβ in ocular posterior segment was sought. METHODS Immunohistochemical localisation of ORβ and ORα protein and detection of OR mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) were performed in macular and extramacular regions of the retina and in the choroid on male and female donors eyes. RESULTS ORβ protein was localised in the ganglion cell layer and in the choroid. At the transcriptional level, mRNA for ORβ and for ORα were both present. Local differences in the expression level were observed, however, suggesting the possibility of variation in the ratio of ORαv ORβ. CONCLUSIONS The coexistence of two oestrogen receptor subtypes in the human ocular posterior segment raises acute questions about their potential physiological role, but offers a perspective for preferential targeting of a specific receptor subtype.

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.

Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor

BACKGROUND The cytokine/chemokine levels of individual follicular fluids (FFs) were measured to determine whether a biomarker could be linked to the developmental potential of the derived embryo. METHODS Fluid was collected from 132 individual FFs that were the source of oocytes subsequently fertilized and transferred. In each, a bead-based multiplex sandwich immunoassay (Luminex) was used to measure 28 cytokines and chemokines simultaneously. RESULTS Significantly higher levels of interleukin (IL-2) and interferon (IFN-gamma) were detected in FF for embryos that underwent early cleavage. IL-12 was significantly higher in FF corresponding to highly fragmented embryos and the chemokine CCL5 was significantly higher in FF related to the best quality (Top) embryos. The level of granulocyte colony-stimulating factor (G-CSF) in individual FF samples was correlated with the implantation potential of the corresponding embryo. The area under the receiver operating characteristics curve, which distinguished the embryos that definitely led to delivery from those that did not, was 0.84 (0.75-0.90) (P = 0.0001) for FF G-CSF. FF G-CSF was significantly lower in patients older than 36 years compared with those <30-year old. When the FF G-CSF was 20 pg/ml or higher, the ratio between Top and non-Top embryos was significantly higher than for the group with FF G-CSF below 20 pg/ml (45 versus 20.45%, P = 0.007). CONCLUSIONS Individual FF composition is related to the development of the corresponding in vitro generated embryo and its potential of implantation. Individual FF G-CSF may provide a non-invasive biomarker of implantation that needs to be evaluated together with in vitro observation to select the oocyte, and hence the embryo, to transfer.