Agnès Noël

Involvement of PA/plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation

© 1997 Federation of European Biochemical Societies.

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)

The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent

Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (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.

Proteases, Extracellular Matrix, and Cancer: A Workshop of the Path B Study Section

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.

MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8)

Matrix metalloproteinases (MMPs) have been implicated in numerous tissue‐remodeling processes. The finding that mice deficient in collagenase‐2 (MMP‐8) are more susceptible to develop skin cancer, prompted us to investigate the role of this protease in cutaneous wound healing. We have observed a significant delay in wound closure in MMP8−/− mice and an altered inflammatory response in their wounds, with a delay of neutrophil infiltration during the first days and a persistent inflammation at later time points. These changes were accompanied by alterations in the TGF‐β1 signaling pathway and by an apoptosis defect in MMP8−/− mice. The delay in wound healing observed in MMP8−/− mice was rescued by bone marrow transplantation from wild‐type mice. Analysis of other MMPs showed that MMP8−/−mice had a significant increase in the expression of MMP‐9, suggesting that both proteases might act coordi‐nately in this process. This possibility was further supported by the novel finding that MMP‐8 and MMP‐9 form specific complexes in vivo. Taken together, these data indicate that MMP‐8 participates in wound repair by contributing to the resolution of inflammation and open the possibility to develop new strategies for treating wound healing defects.—Gutierrez‐Fernandez, A., Inada, M., Balbín, M., Fueyo, A., Pitiot, A. S., Astudillo, A., Hirose, K., Hirata, M., Shapiro, S. D., Noel, A., Werb, Z., Krane, S. M. Lopez‐Otín, C., Puente, X. S. FASEB J. 21, 2580–2591 (2007)

Vascular endothelial growth factor expression correlates with matrix metalloproteinases MT1-MMP, MMP-2 and MMP-9 in human glioblastomas.

Vascular endothelial growth factor (VEGF) is the major endothelial mitogen in central nervous system neoplasms and it is expressed in 64–95% of glioblastomas (GBMs). Tumour cells are the main source of VEGF in GBMs whereas VEGF receptors (VEGFR‐1, its soluble form sVEGFR‐1, VEGFR‐2 and neuropilin‐1) are expressed predominantly by endothelial cells. Infiltrating tumour cells and newly‐formed capillaries progress through the extracellular matrix by local proteolysis involving matrix metalloproteinases (MMPs). Recent studies have shown that VEGF expression and bioavailability can be modulated by MMPs. We reported previously that the expression of MT1‐MMP in human breast cancer cells was associated with an enhanced VEGF expression. We used quantitative RT‐PCR, Western blot, gelatin zymography and immunohistochemistry to study the expression of VEGF, VEGFR‐1, VEGFR‐2, sVEGFR‐1, neuropilin‐1, MT1‐MMP, MMP‐2, MMP‐9 and TIMP‐2 in 20 human GBMs and 5 normal brains. The expression of these MMPs was markedly increased in most GBMs with excellent correlation between mRNA and protein levels; activated forms of MMP‐2 and MMP‐9 were present in 8/18 and 7/18 of GBMs. A majority of GBMs (17/20) also expressed high levels of VEGF, as previously reported, with strong correlation between VEGF and MT1‐MMP gene expression levels, and double immunostaining showed that VEGF and MT1‐MMP peptides co‐localize in tumour and endothelial cells. Our results suggest that the interplay between metalloproteinases and VEGF previously described in experimental tumours may also be operative in human GBMs. Because of its dual ability to activate MMP‐2 and to up‐regulate VEGF, MT1‐MMP might be of central importance in the growth of GBMs and represent an interesting target for anti‐cancer treatments.

MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells

Background MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated. Methodology/Principal Findings We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization. Conclusions/Significance Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.