Carine Munaut

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.

High level of MT‐MMP expression is associated with invasiveness of cervical cancer cells

MMP‐2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane‐associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF‐β, but its physiological regulation is still largely unresolved. MT‐MMP was recently discovered and described as a potential gelatinase‐A activator. In the present study, we investigated the expression of MT‐MMP (membrane‐type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro‐transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT‐MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT‐MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non‐invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT‐MMP. Taken together, our results clearly correlated high level MT‐MMP expression with invasiveness, and thus suggested that MT‐MMP might play a crucial role in cervical tumor invasion.

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.

Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti‐ or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP‐9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP‐2 and MMP‐3 were stromal. Neither the single deficiency of MMP‐2, MMP‐3, or MMP‐9, nor the combined absence of MMP‐9 and MMP‐3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP‐2:MMP‐9‐deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP‐2 and MMP‐9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP‐2 and MMP‐9. Our data demonstrate for the first time in an experimental model that MMP‐2 and MMP‐9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.

Matrix metalloproteinase-9 deficiency impairs cellular infiltration and bronchial hyperresponsiveness during allergen-induced airway inflammation.

We investigated the specific role of matrix metalloproteinase (MMP)-9 in allergic asthma using a murine model of allergen-induced airway inflammation and airway hyperresponsiveness in MMP-9(-/-) mice and their corresponding wild-type (WT) littermates. After a single intraperitoneal sensitization to ovalbumin, the mice were exposed daily either to ovalbumin (1%) or phosphate-buffered saline aerosols from days 14 to 21. Significantly less peribronchial mononuclear cell infiltration of the airways and less lymphocytes in the bronchoalveolar lavage fluid were detected in challenged MMP-9(-/-) as compared to WT mice. In contrast, comparable numbers of bronchoalveolar lavage fluid eosinophils were observed in both genotypes. After allergen exposure, the WT mice developed a significant airway hyperresponsiveness to carbachol whereas the MMP-9(-/-) mice failed to do so. Allergen exposure induced an increase of MMP-9-related gelatinolytic activity in WT lung extracts. Quantitative reverse transcriptase-polymerase chain reaction showed increased mRNA levels of MMP-12, MMP-14, and urokinase-type plasminogen activator after allergen exposure in the lung extracts of WT mice but not in MMP-9-deficient mice. In contrast, the expression of tissue inhibitor of metalloproteinases-1 was enhanced after allergen exposure in both groups. We conclude that MMP-9 plays a key role in the development of airway inflammation after allergen exposure.

MMP-2 and MMP-9 synergize in promoting choroidal neovascularization

Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age‐related macular degeneration (AMD), but their precise role and potential interactions remain unclear. To address the question of MMP‐2 and MMP‐9 functions, mice deficient in the expression of MMP‐ 2 (MMP‐2 KO), MMP‐9 (MMP‐9 KO), and both MMP‐2 and MMP‐9 (MMP‐2,9 KO) with their corresponding wild‐type mice (WT) underwent CNV induction by laser‐induced rupture of the Bruchs membrane. Both the incidence and the severity of CNV were strongly attenuated in double deficient compared with single gene deficient mice or corresponding WT controls. The reduced neovascularization was accompanied by fibrinogen/fibrin accumulation. Furthermore, overexpression of the endogenous MMP inhibitors TIMP‐1 or TIMP‐2 (delivered by adenoviral vectors) in WT mice or daily injection of a synthetic and gelatinase selective MMP inhibitor (Ro 26‐2853) significantly decreased the pathological reaction. These findings suggest that MMP‐2 and MMP‐9 may cooperate in the development of AMD and that their selective inhibition represents an alternative strategy for the treatment of choroidal neovascularization.

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.

Smooth muscle cell matrix metalloproteinases in idiopathic pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) results from persistent vasoconstriction, smooth muscle growth and extracellular matrix (ECM) remodelling of pulmonary arteries (PAs). Matrix metalloproteinases (MMPs) are matrix-degrading enzymes involved in ECM turnover, and in smooth muscle cell (SMC) and endothelial cell migration and proliferation. MMP expression and activity are increased in experimental PAH. Therefore, this study investigated whether similar changes occur in idiopathic PAH (IPAH; formerly known as primary pulmonary hypertension). Both in situ and in vitro studies were performed on PAs from patients undergoing lung transplantation for IPAH and from patients treated by lobectomy for localised lung cancer, who served as controls. In IPAH, MMP–tissue inhibitor of metalloproteinase (TIMP) imbalance was found in cultured PA-SMCs, with increased TIMP-1 and decreased MMP-3. MMP-2 activity was markedly elevated as a result of increases in both total MMP-2 and proportion of active MMP-2. In situ zymography and immunolocalisation showed that MMP-2 was associated with SMCs and elastic fibres, and also confirmed the MMP-3–TIMP-1 imbalance. In conclusion, the findings of this study were consistent with a role for the matrix metalloproteinase–tissue inhibitor of metalloproteinase system in pulmonary vascular remodelling in idiopathic pulmonary arterial hypertension. The matrix metalloproteinase–tissue inhibitor of metalloproteinase imbalance may lead to matrix accumulation, and increased matrix metalloproteinase-2 activity may contribute to smooth muscle cell migration and proliferation. Whether these abnormalities are potential therapeutic targets deserves further investigation.

Dysregulation of anti-angiogenic agents (sFlt-1, PLGF, and sEndoglin) in preeclampsia—a step forward but not the definitive answer

Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, which resolves on placental delivery. It is thought to be the consequence of impaired placentation due to inadequate trophoblastic invasion of the maternal spiral arteries. In PE the maternal plasma concentration of free vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) is decreased whereas the concentration of soluble fms-like tyrosine kinase-1 (sFlt-1) and of soluble endoglin (sEng) is increased. These soluble receptors may bind VEGF, PLGF and TGFbeta1 and TGFbeta3 in the maternal circulation, causing endothelial dysfunction in many maternal tissues. Hence there is a view that the pathogenesis is more or less clarified. According to the vascular theory, poor placentation leads to poor uteroplacental perfusion and hypoxia, which stimulates sFlt-1 and sEng production causing the maternal syndrome. This assumption has been recently challenged. The role of hypoxia as the main stimulus for release of sFlt-1 has been questioned and the role of inflammatory mechanisms has been emphasized. According to this inflammatory theory, poor placentation may predispose more to placental oxidative stress than hypoxia and endothelial dysfunction may be part of a broader disorder of systemic inflammation. Finally, the recent demonstration of activating auto-antibodies to the angiotensin 1 receptor that experimentally play a major pathogenic role in PE further suggests a pleiotropism of aetiologies for this condition. The purpose of this review is to critically evaluate the recent hypotheses and their possible insights on early diagnosis, prevention and treatment.