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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5828-5836
Copyright © 2004 by The Endocrine Society

Involvement of Membrane-Type Matrix Metalloproteinases (MT-MMPs) in Capillary Tube Formation by Human Endometrial Microvascular Endothelial Cells: Role of MT3-MMP

Margreet Plaisier, Kitty Kapiteijn, Pieter Koolwijk, Catherine Fijten, Roeland Hanemaaijer, Jos M. Grimbergen, Adri Mulder-Stapel, Paul H. A. Quax, Frans M. Helmerhorst and Victor W. M. van Hinsbergh

Department of Biomedical Research (M.P., K.K., P.K., C.F., R.H., J.M.G., A.M.-S., P.H.A.Q., V.W.M.v.H.), Gaubius Laboratory TNO Prevention and Health, 2301 CE, Leiden, The Netherlands; Department of Gynecology and Reproductive Medicine (M.P., K.K., F.M.H.), Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; and Department of Physiology (V.W.M.v.H.), Institute for Cardiovascular Research, VU University Medical Center, 1081 BT, Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: V. W. M. van Hinsbergh, Ph.D., Gaubius Laboratory TNO-PG, Zernikedreef 9, 2333 CK Leiden, The Netherlands. E-mail: v.vanhinsbergh{at}vumc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the endometrium, angiogenesis is a physiological process, whereas in most adult tissues neovascularization is initiated only during tissue repair or pathological conditions. Pericellular proteolysis plays an important role in angiogenesis being required for endothelial cell migration, invasion, and tube formation. We studied the expression of proteases by human endometrial microvascular endothelial cells (hEMVECs) and their involvement in the formation of capillary tubes and compared these requirements with those of foreskin MVECs (hFMVECs). Inhibition of urokinase and matrix metalloproteinase (MMP) both reduced tube formation in a fibrin or fibrin/collagen matrix. hEMVECs expressed various MMP mRNAs and proteins; in particular MMP-1, MMP-2, and membrane-type (MT)1-, MT3-, and MT4-MMPs. MT3- and MT4-MMP mRNA expressions were significantly higher in hEMVECs than in hFMVECs. Other MT-MMP mRNAs and MMP-9 were hardly detectable. Immunohistochemistry confirmed the presence of MT3-MMP in endothelial cells of endometrial tissue. Overexpression of tissue inhibitor of MMP (TIMP)-1 or TIMP-3 by adenoviral transduction of hEMVECs reduced tube formation to the same extent, whereas only TIMP-3 was able to inhibit tube formation by hFMVECs. Tube formation by hEMVECs was partly inhibited by the presence of anti-MT3-MMP IgG. Thus, in contrast to tube formation by hFMVECs, which largely depends on MT1-MMP, capillary-like tube formation by hEMVECs is, at least in part, regulated by MT3-MMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ADULT, angiogenesis plays a role in many pathological conditions, such as the growth of solid tumors, diabetic retinopathy, rheumatoid arthritis, and wound healing (1, 2). Physiological angiogenesis during adulthood is limited to the female reproductive tissue, namely in the ovary and endometrium. Endometrial angiogenesis plays a role in endometrial remodeling during the menstrual cycle and after conception during the implantation of the embryo (3, 4, 5). Angiogenesis is initiated by a shift in the balance between proangiogenic and antiangiogenic factors (6, 7). It involves the sprouting of new capillary-like structures from existing vasculature and may involve blood-borne cells that intussuscepts in and around the new vascular structures (2). These newly formed tubes are subsequently stabilized, often by interaction with pericytes. Although the general mechanisms of angiogenesis are probably rather similar in various tissues, the individual players, such as growth factors, integrins, and proteases, may vary in different tissues. Endothelial cells from different tissues and vessel types have specific properties (8), many of which are conserved in vitro (8, 9, 10). We previously observed that different types of human microvascular endothelial cells (hMVECs) have different requirements for proliferation and capillary tube formation in vitro. Although endometrial MVECs (hEMVECs) are highly sensitive to vascular endothelial growth factor (VEGF)-A and form capillary tubules after exposure to VEGF-A (9), foreskin MVECs (hFMVECs) are more sensitive to basic fibroblast growth factor (bFGF) and form capillary tubes in a fibrin matrix only after simultaneous exposure to bFGF or VEGF-A and the inflammatory cytokine TNF{alpha} (11, 12).

Among the various processes that regulate angiogenesis, the generation of proteolytic activity is thought to be pivotal in the regulation of cell migration and capillary tube formation (13). Key regulators of pericellular proteolysis and capillary-like tubule formation by endothelial cells are cell-bound urokinase-type plasminogen activator (u-PA) and plasmin as well as matrix metalloproteinases (MMPs) (5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Initial data on the formation of tubular structures by hEMVECs indicated that cell-bound u-PA and plasmin contribute to this process (9). In addition to the u-PA/plasmin cascade, the rapidly expanding family of MMPs (21) plays an important role in cell migration and invasion, and in angiogenesis in vivo (19, 22, 23). MMPs are widely expressed in the endometrium and play a role in tissue degradation and menstrual bleeding (24). Furthermore, a number of them are also detected during the proliferative and early secretory phase (25), which suggests a role in endometrial remodeling and angiogenesis (26, 27). However, the exact role of MMPs in endometrial angiogenesis in vivo and tube formation by hEMVECs in vitro is unknown.

Membrane-type (MT)-MMPs have been suggested to play a key role in angiogenesis, in addition to the gelatinases MMP-2 and -9 (17, 28, 29). The membrane-associated localization of MT-MMPs makes this group of MMPs particularly suited to function in pericellular proteolysis (17). Six MT-MMPs have been described: four transmembrane proteins and two glycosylphosphatidylinositol (GPI)-anchored ones. Recently, MT1-MMP (MMP-14) received considerable attention as being involved in endothelial cell migration and invasion (14, 15, 16). MT1-MMP contributes to angiogenesis by its capacity to degrade extracellular matrix components, thereby promoting cell migration, invasion, and possibly the bioavailability of growth factors. Furthermore, it activates pro-MMP-2 [via the tissue inhibitor of MMP (TIMP)-2-MT1-MMP complex], pro-MMP-13, and {alpha}vß3-integrin, an important integrin in angiogenesis (14, 29, 30, 31). MT1-MMP as well as MMP-2 are able to stimulate angiogenesis (32, 33). In hFMVECs, MT1-MMP becomes a key factor in capillary tube formation when collagen is present in the fibrinous matrix (16, 17). MT2-MMP (MMP-15) and MT3-MMP (MMP-16) are also involved in cell migration and invasion, depending on the cell type (17, 34). Their overexpression in endothelial cells can induce capillary-tube formation, similar to MT1-MMP (28). MT1-MMP and MT2-MMP are present in endometrial tissue during various stages of the menstrual cycle; MT3-MMP mRNA is increased during the proliferative phase of the endometrium (35, 36, 37, 38). It is generally believed that these MMPs also play a role in endometrial angiogenesis (39), but except for the expression and immunolocalization of specific MMPs in endometrial tissue little information is available.

The activity of MMPs and MT-MMPs is regulated by activation of the proenzymes and by specific inhibitors, the TIMPs and {alpha}-macroglobulins. The TIMP family consists of four members, which differ in expression patterns, regulation, and ability to interact specifically with latent MMPs and members of the related metalloproteinases of the family of metalloproteinases with a disintegrin and a metalloproteinase domain and TNF{alpha} converting enzyme group (40). TIMP-1 is secreted as a soluble protein and has a general inhibiting activity on many MMPs but does not inhibit MT1-MMP. TIMP-3 is associated with the matrix components and has a similar inhibitory spectrum but also inhibits MT1-MMP (41). Furthermore, TIMP-3 can induce apoptosis in various cell types (40).

In this study we report on the expression of MMPs and MT-MMPs by hEMVECs and the requirement of these proteases for capillary-like tube formation by these cells. By overexpressing TIMP-1 and TIMP-3 we could demonstrate that different MMPs act as key regulators for tube formation by hEMVECs and hFMVECs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Penicillin/streptomycin, L-glutamine, and tissue culture medium 199 (M199) with 20 mM HEPES with or without phenol red were obtained from BioWhittaker (Verviers, Belgium). Newborn calf serum (NBCS) was obtained from Life Technologies (Grand Island, NY). Human serum (HS), prepared from fresh blood from 10–20 healthy donors, was obtained from a local blood bank and was pooled and stored at 4 C. NBCS and HS were heat-inactivated before use. Pyrogen-free HS albumin (HSA) was obtained from Sanquin (Amsterdam, The Netherlands). Tissue culture plastics and microtiter plates were obtained from Costar/Corning (Cambridge, MA) and Falcon (Becton Dickinson Biosciences, Bedford, MA). A crude preparation of endothelial cell growth factor (ECGF) was prepared from bovine brain as described by Maciag et al. (42). Heparin and thrombin were obtained from Leo Pharmaceutics Products (Weesp, The Netherlands). Human fibrinogen was obtained from Chromogenics AB (Mölndal, Sweden). Dr. H. Metzner and Dr. G. Seeman (Aventis Behring GmbH, Marburg, Germany) generously provided factor XIII. Fibronectin was a gift from Dr. J. van Mourik (CLB, Amsterdam, The Netherlands). Rat tail collagen type-I was obtained from Becton Dickinson Biosciences. Human recombinant VEGF-A was obtained from RELIATech (Braunschweig, Germany), and TNF{alpha} was a gift from Dr. J. Travernier (Biogent, Gent, Belgium). Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma Chemical Co. (St. Louis, MO). Adenoviral vectors containing LacZ, TIMP-1, and TIMP-3 were previously described (43, 44, 45). Aprotinin was purchased from Pentapharm Ltd (Basel, Switzerland). BB94 (Batimastat) was a kind gift from Dr. E.A. Bone (British Biotech, Oxford, UK). Rabbit antihuman polyclonal antibodies against u-PA, MMP-9, and MT1-MMP were produced and characterized in our laboratory (11, 16, 46, 47). Mouse antihuman monoclonal antibody against MT3-MMP was obtained from Oncogene Research Products (IM50L; Boston, MA), biotinylated horse antimouse antibody from Vector Laboratories (BA-2000; Burlingame, CA), avidin-biotin complex from DakoCytomation (Glostrup, Denmark), and NovaRED from Vector. Human recombinant MT1-MMP (pro-domain-catalytic domain-hemopexin domain) was purchased from Chemicon (Temecula, CA), and recombinant pro-MMP-9 was from Invitek (Berlin, Germany). PBS/T concentrate was obtained from Organon Teknika (Boxtel, The Netherlands). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control reagents (VIC labeled) were purchased from Applied Biosystems (Nieuwerkerk a/d/ IJssel, The Netherlands). For Western blotting, protease inhibitors from Roche Diagnostics, Immobilon-P polyvinylidene fluoride transfer membranes from Millipore (Bedford, MA), skim milk powder from Merck (Amsterdam, The Netherlands), goat anti-ß-actin antibody (sc-1615), and horseradish peroxidase-conjugated secondary antibodies from Santa Cruz (Heerhugowaard, The Netherlands) were used. The Super Signal West Dura extended duration substrate purchased from Pierce (St. Augustin, Germany) and the luminescent image workstation from Roche Diagnostics (Almere, The Netherlands) were used for visualization.

Cells

hEMVECs were isolated, cultured, and characterized as previously described in detail (9). In short, endometrial tissue was obtained from premenopausal women who underwent uterus extirpation for benign pathology. The tissue was collected according to the guidelines of the Institutional Review Board, and informed consent was obtained from each patient. Endometrial tissue was scraped from the uterus and stored overnight at 4 C. The following day, tissue was minced and cells extracted using 0.2% collagenase. The primary heterogeneous culture was purified by repeated selections using anti-CD31 and anti-IgG-coated Dynabeads. After purification of the culture, the endothelial cells were characterized as being positive for CD31 and von Willebrand factor and negative for cytokeratin-18 and {alpha}-smooth muscle actin. hEMVECs were maintained in hEMVEC culture medium: M199 without phenol-red supplemented with 20 mM HEPES (pH 7.3), 20% HS, 10% NBCS, 150 µg/ml ECGF, 5 U/ml heparin, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The cells were cultured on fibronectin-coated dishes under humidified 5% CO2/95% air atmosphere. VEGF-A (5 ng/ml) was added to the culture medium of the primary isolates to facilitate the initial growth of the endothelial cells. Endometrial tissues were obtained from all phases of the menstrual cycle, as determined by histological dating according to Noyes et al. (48), and hEMVECs from different stages showed comparable functions in vitro.

hFMVECs were isolated, characterized, and cultured as previously described (10, 49).

In vitro capillary-like tube formation assay

Human fibrin matrices were prepared as described before (9). For the collagen gels, 7 vol of rat tail collagen type-I (3 mg/ml) were mixed with 1 vol of 10x M199 with phenol red and 2 vol of 2% (wt/vol) Na2CO3 (final pH 7.4), and 300-µl aliquots were added to each well of a 48-well plate and allowed to gelate at 37 C in the absence of CO2.

Confluent hEMVECs were detached and seeded at a split ratio of 2:1 on top of the fibrin and/or collagen matrices and cultured for 24 h in hEMVEC culture medium without ECGF and heparin. Subsequently, the endothelial cells were cultured with the mediators indicated for 2–5 d. Invading cells and the formation of capillary-like structures of endothelial cells in the three-dimensional fibrin and/or collagen matrix were analyzed by phase contrast microscopy. The total length of the structures formed was measured in six randomly chosen microscopic fields (7.3 mm2/field) by computer-equipped Optimas image analysis software (Bioscan, Demons, WA) connected to a monochrome CCD camera (MX5) and expressed as mm/cm2 (7, 24).

Gelatin zymography

Gelatinolytic activities of MMPs secreted by hEMVECs were analyzed by zymography on gelatin-containing polyacrylamide gels as described (50). Using this technique, we can visualize both active and latent species. Samples were applied to a 10% (wt/vol) acrylamide gel copolymerized with 0.2% (wt/vol) gelatin. After electrophoresis, the gels were washed three times for 10 min in 50 mmol/liter Tris/HCl (pH 8.0) containing 5 mmol/liter CaCl2, 1 µmol/liter ZnCl2, and 2.5% (wt/vol) Triton X-100 to remove the SDS, followed by three washes of 5 min in 50 mmol/liter Tris/HCl (pH 8.0) containing 5 mmol/liter CaCl2 and 1 µmol/liter ZnCl2 and incubated overnight at 37 C. The gels were stained with Coomassie Brilliant Blue R-250.

Immunohistochemistry

Immunohistochemical staining of MT3-MMP was performed in paraffin-embedded sections of human endometrium. Sections were deparaffinized and endogenous peroxidase was quenched with 3% H2O2 in 100% methanol. To prevent nonspecific binding, sections were incubated with 5% BSA for 15 min. The primary monoclonal mouse anti-MT3-MMP antibody (1 µg/ml in 1% BSA in PBS) was applied overnight at 4 C, followed by a 1-h incubation with a biotinylated secondary horse antimouse antibody (5 µg/ml in 1% BSA/PBS). Streptavidin-horseradish peroxidase conjugate was used to obtain red staining of the antigens. Specificity of the immunohistochemical reaction was verified by omission of the first antibody as well as using normal mouse serum instead of the first antibody. Sections were counterstained with Mayer’s hematoxylin.

Western blotting

Total cellular extracts were prepared in the presence of protease inhibitors and applied to SDS-PAGE electrophoresis essentially as described (51). After proteins were blotted onto Immobilon-P polyvinylidene fluoride transfer membranes, the blots were blocked with 5% (wt/vol) skim milk powder diluted in 20 mM Tris (pH 7.4), 55 mM NaCl, and 0.1% (vol/vol) Tween 20. Then, blots were incubated with a mouse anti-MT3-MMP antibody or a goat anti-ß-actin antibody followed by horseradish peroxidase-conjugated secondary antibodies. All antibodies were diluted in 20 mM Tris (pH 7.4), 55 mM NaCl, 0.1% (vol/vol) Tween 20, and 5% (wt/wt) bovine serum. The Super Signal West Dura extended duration substrate and the luminescent image workstation were used for visualization.

RNA isolation and real-time RT-PCR

Total RNA from hEMVECs and hFMVECs was isolated as described by Chomczynski and Sacchi (52). RNA was quantified by measuring its absorbance using a spectrophotometer and considered of good quality when the OD260/OD280 ratio ranged between 1.8 and 2.0. RT was carried out in 20-µl vol using random primers and a cDNA synthesis kit purchased from Promega. MMP and MT-MMP expression was quantified using real-time PCR according to the TaqMan method of Applied Biosystems (Perkin-Elmer, Norwalk, CT) using a forward and reverse primer combined with a specific (6-carboxy-fluorescein/6-carboxy-tetramethyl-rhodamine) double-labeled probe. The following sequences were used for MT3-MMP (MMP-16): forward primer, 5'-GGC TCG TGT GGG AAA TGG TA-3'; reverse primer, 5'-AGA ACT CTT CCC CCT CAA GTG-3'; and probe, 5'-ACA GCT GGC TCT ACT TCC CCA TGG C-3'. Primers and probes for MT1-MMP were described previously (16). All data were controlled for quantity of RNA input by performing measurements on the endogenous reference gene GAPDH (VIC-labeled) as follows. For each RNA sample, a difference in number of cycles (CT) values (dCT) was calculated for each mRNA by taking the mean CT of duplicate wells and subtracting the mean CT of the duplicate wells for the reference RNA GAPDH measured in the same RT reaction. All RT reactions were carried out in quadruplicate. The following were used as positive controls: cDNA of human endometrial stromal cells for MMP-12, cDNA of HT1080 cells for MMP-13, and double-stranded cDNA encoding for MMP-3, MMP-7, and MMP-8.

Adenoviral gene transfer of TIMP-1 and TIMP-3 to hEMVECs and hFMVECs

Replication-deficient adenoviral vectors (E1-deleted, transcriptional control via the cytomegalovirus promoter) encoding human TIMP-1 (AdTIMP-1), human TIMP-3 (AdTIMP-3), and a ß-galactosidase-encoding adenoviral vector (AdLacZ), as a control, were used for the experiments (43). Confluent hEMVECs and hFMVECs were washed twice with M199 supplemented with 0.1% HSA to remove HS components. Subsequently, the hEMVECs were incubated with the adenoviral constructs in M199 containing 0.1% HSA for 2 h. After transduction, the medium was replaced with hEMVEC culture medium without VEGF-A. Twenty-four hours later the cells were seeded on top of a three-dimensional fibrin/fibrin-collagen matrix and stimulation was started 6 h after seeding.

TIMP-1 ELISA and MMP bioactivity assays

TIMP-1 antigen was assayed by ELISA (R&D Systems, Oxon, UK). MT1-MMP and MMP-9 activity were determined by MMP activity assays (Biotrak; Amersham Biosciences, Little Chalfont, UK) as previously indicated (16, 46). Selective TIMP-3 activity over that of TIMP-1 was assayed by determination of active MT1-MMP in extracts of hEMVECs transduced with AdLacZ, AdTIMP-1, and AdTIMP-3. Inhibition of MMP-9 activity by TIMP-1 and TIMP-3 was determined by addition of serial dilutions of 48-h conditioned media of hEMVECs transduced with AdLacZ, AdTIMP-1, and AdTIMP-3 to 4-aminophenylmercuric acetate-activated recombinant pro-MMP-9.

Statistics

Experiments were performed with duplicate wells and expressed as mean ± SEM. For statistical evaluation, the ANOVA was used, followed by a modified t test according to Bonferroni. Statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Capillary-like tube formation by hEMVECs is inhibited by collagen type-I

Three-dimensional matrices were prepared consisting of pure fibrin, collagen, or mixtures of fibrin and collagen. As previously reported (9), hEMVECs form spontaneously capillary-like tubular structures in a fibrin matrix, a process that is markedly enhanced by VEGF-A (Fig. 1Go, A and C). When hEMVECs were seeded on top of matrices containing 0–50% type-I collagen homogeneously mixed with fibrin, a concentration-dependent decrease in the extent of tube formation was seen. In a mixed collagen-fibrin matrix (50/50), the decrease was 55 ± 4% under basal conditions (n = 3, not shown) and 53 ± 2% in the presence of VEGF-A (Fig. 1BGo) compared with the tube formation in a pure fibrin matrix (Fig. 1AGo). In a pure collagen type-I matrix, capillary-like structure formation by hEMVECs was hardly detectable, even after stimulation with VEGF-A (data not shown).



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  		FIG. 1. Capillary-like tube formation by hEMVECs in a fibrin or collagen matrix depends on u-PA and MMP activities. hEMVECs were cultured on top of a three-dimensional fibrin matrix (A, C, and D) or 50/50% fibrin/collagen-type-1 matrix (B and E) and stimulated with VEGF-A (10 ng/ml). A and B, Micrographs taken after 4 d of culturing; insets in A and B show details of capillary-like structures. Bars, 300 µm; inset bars, 100 µm. C, Cross-section perpendicular to the matrix surface and stained with hematoxylin-phloxine- safran. Bar, 50 µm. D and E, hEMVECs were cultured with 10 ng/ml VEGF-A (control) in the absence or presence of polyclonal anti-u-PA ({alpha}uPA, 100 µg/ml), BB94 (5 µg/ml), or a combination of BB94 and anti-u-PA. After 3–5 d of culturing, mean tube length was measured by image analysis. The data in D are expressed as a percentage of VEGF-A-induced tube formation ± SEM of six independent experiments of duplicate wells performed with three different hEMVEC isolations. E, Three experiments are represented. *, P < 0.05 vs. control, #, P < 0.05 vs. {alpha}uPA.

 
u-PA/plasmin and MMPs are involved in tube formation by hEMVECs in matrices composed of fibrin and/or collagen

To establish the involvement of u-PA/plasmin and MMPs in the formation of capillary-like structures by hEMVECs, u-PA-blocking antibodies, the plasmin inhibitor aprotinin, or the broadly acting metalloproteinase inhibitor BB94 were used (Fig. 1Go, C and D). The VEGF-A-enhanced tube formation in a fibrin matrix was reduced by 55 ± 11% by u-PA-blocking antibodies (Fig. 1DGo) and by 54 ± 7% by the plasmin inhibitor aprotinin (data not shown). In a matrix consisting of an equal mixture of fibrin and collagen, anti-u-PA antibodies reduced tube formation only by 17 ± 0% (Fig. 1EGo). The inhibiting effect of BB94 was increased by adding collagen, because tube formation in pure fibrin was inhibited by 31 ± 5% and in collagen-fibrin matrices by 64 ± 3%. An almost complete inhibition (84 ± 6% and 82 ± 2%, respectively) of capillary-like structure formation was seen after the simultaneous addition of BB94 and anti-u-PA antibodies (Fig. 1Go, D and E).

hEMVECs express various MMPs and MT-MMPs

To study which MMPs are expressed by hEMVECs, real-time RT-PCR was used to assess the expression and regulation of MMP mRNA levels in hEMVECs. Real-time RT-PCR revealed that hEMVECs expressed considerable amounts of MMP-1, MMP-2, MT1-MMP, MT3-MMP, and MT4-MMP mRNAs (i.e. less than 30 cycles and dCT < 9) under basal as well as VEGF-A-stimulated conditions. The data for the MT-MMPs are given in Table 1Go. hFMVECs had a similar expression pattern as hEMVECs, except for MMP-1, which was poorly expressed by hFMVECs under basal conditions (not shown), and MT3-MMP and MT4-MMP, which were expressed to a higher degree in hEMVECs (Table 1Go). Under basal and VEGF-A-stimulated conditions, hEMVECs expressed relatively small amounts of MMP-9 [mean CT = 35.3 ± 1.5 cycles; mean dCT = 14.4 ± 1.3 (± SEM)] and MT2-, MT5-, and MT6-MMP (Table 1Go). The MMP-9 mRNA expression increased markedly when the cells were stimulated with 10–8 M phorbol ester PMA [mean CT = 27.4 ± 1.0; dCT = 8.4 ± 0.7 (± SEM)]. No mRNA of MMP-3, MMP-7, MMP-8, MMP-12, and MMP-13 was detected in hEMVECs. Positive controls resulted in abundant signals: double-stranded cDNA encoding for MMP-3, MMP-7, and MMP-8; cDNA of human endometrial stromal cells for MMP-12; and cDNA of HT1080 cells for MMP-13.


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  		TABLE 1. Analysis of MT-MMP mRNA expression in VEGF-A-stimulated hEMVEC, and hFMVECs

 
The expression of active MMPs was confirmed by gelatin zymography and activity assays. Gelatin zymography of serum-free hEMVEC-conditioned media (24 h) showed expression of latent MMP-2 (72 kDa) and a 55-kDa band that represents MMP-1 or MMP-3. From the mRNA data we assume that the 55-kDa band represents MMP-1 rather than MMP-3. Stimulation with 10–8 M PMA induced MMP-9 (92 kDa) protein synthesis and activation of MMP-2 (64 kDa; Fig. 2AGo). The presence of MT1-MMP was demonstrated by activity assay (Fig. 2BGo). Both VEGF-A and TNF{alpha} exposure doubled the activity of MT1-MMP, whereas phorbol ester caused a dramatic increase in MT1-MMP activity in hEMVECs (Fig. 2BGo). The presence of MT3-MMP protein was confirmed by Western blotting. The production of MT3-MMP was not affected by TNF{alpha} and increased slightly after VEGF-A exposure (Fig. 2CGo). MT3-MMP was detectable in endothelial cells of proliferative human endometrial tissue as well as in endometrial epithelial cells and myometrial cells (Fig. 2Go, D and E).



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  		FIG. 2. hEMVECs express various MMPs and MT-MMPs. hEMVECs were cultured for 24 h in M199 supplemented with 0.5% HSA (A) or 20% HS (B and C) and were not stimulated (control) or stimulated with TNF{alpha} (2.5 ng/ml), VEGF-A (10 ng/ml), or PMA (10–8 M), as indicated. A, Gelatin zymography of 24 h conditioned medium (M, ladder); B, MT1-MMP activity in cell lysates (mean ± range of two experiments performed in duplicate wells with two different isolations; detection limit of the assay, 0.2 ng/ml); C, Western blot of MT3-MMP in 24-h conditioned medium; D and E, immunohistochemical analysis of MT3-MMP in endometrial tissue shows the presence of MT3-MMP in endothelial cells (D, arrows) and myometrium (E, stars). Similar results were obtained in the tissue of three other donors.

 
Adenoviral gene transfer of both TIMP-1 and TIMP-3 impairs VEGF-A-induced tube formation by hEMVECs

Because the general metalloproteinase inhibitor BB94 inhibited tube formation by hEMVECs, the effects of TIMP-1 and TIMP-3, two physiological tissue inhibitors of MMPs, on this process were studied. hEMVECs were infected for 2 h with replication-deficient adenoviruses expressing human TIMP-1 (AdTIMP-1), TIMP-3 (AdTIMP-3), or a control LacZ (AdLacZ). Transduction of hEMVECs with AdTIMP-1 caused a concentration-dependent increase in TIMP-1 antigen production, whereas AdLacZ or AdTIMP-3 did not affect TIMP-1 production (Fig. 3AGo). To verify whether the overexpressed TIMP-1 and -3 were functional and active, their effects on MT1-MMP and MMP-9 activity were analyzed. In contrast to cell extracts of AdLacZ- or AdTIMP-1-transduced hEMVECs, in which MT1-MMP remained active, MT1-MMP activity was completely inhibited in cell extracts of AdTIMP-3-transduced hEMVECs (Fig. 3BGo). AdTIMP-1- and AdTIMP-3-transduced hEMVECs inhibited exogenous active MMP-9 comparably (Fig. 3CGo).



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  		FIG. 3. hEMVECs overexpress active TIMP-1 and -3 antigen after transduction. Confluent hEMVECs were transduced with 1.25 x 106, 2.5 x 106, and 1.0 x 108 pfu/ml AdLacZ, AdTIMP-1, or AdTIMP-3 as described in Materials and Methods. After 2 h, the medium was removed and the cells were incubated for 6 h with hEMVEC culture medium and incubated for 48 h in M199 supplemented with 0.5% HSA and 10 ng/ml VEGF-A with or without PMA (10–8 M). A, TIMP-1 levels were determined in the hEMVEC-conditioned medium by ELISA following the manufacturers’ descriptions; B, MT1-MMP activity in the lysates of PMA-stimulated transduced hEMVECs was inhibited by TIMP-3 and not by TIMP-1. MT1-MMP activity was analyzed as indicated in Materials and Methods. C, The ability of TIMP-1 and TIMP-3 to inhibit MMP-9 activity was measured in 100-fold (black bars) and 25-fold (gray bars) dilutions of the conditioned media of hEMVECs overexpressing TIMP-1 or TIMP-3.

 
Previous studies on human umbilical vein endothelial cells and hFMVECs have shown that TIMP-3 was a more potent inhibitor of capillary-tube formation than TIMP-1 (15, 16, 53). Unexpectedly, in hEMVECs, both TIMP-1 and TIMP-3 overexpression inhibited VEGF-A-induced tube formation, to an extent similar to BB94 (Fig. 4Go, A and B). This was found both in fibrin and in fibrin-collagen matrices (Fig. 4BGo). No apparent cell death or morphological changes were observed in either the AdTIMP-1- or AdTIMP-3-transduced hEMVECs.



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  		FIG. 4. Both TIMP-1 and TIMP-3 inhibit capillary-like tube formation by hEMVECs. hEMVECs were transduced with 2.5 x 106 pfu/ml AdLacZ, AdTIMP-1, and AdTIMP-3 and were cultured on top of a three-dimensional fibrin matrix or a fibrin-10% collagen matrix and stimulated with VEGF-A (10 ng/ml) with or without BB94 (5 µg/ml). A, Phase contrast micrographs after 3 d of culturing showing tube formation in the fibrin matrix. Bar, 300 µm. B, Mean tube length was measured and expressed as a percentage of the tube formation by the AdLacZ-transduced cells ± SEM/range of five (fibrin matrix, black bars) and two (fibrin-collagen matrix, striped bars) independent experiments performed in duplicate wells. The mean tube length of the AdLacZ-transduced hEMVECs was 239 ± 13 mm/cm2 on the fibrin-collagen matrix. *, P < 0.03 vs. LacZ-transduced cells.

 
Comparison of the effect of TIMP-1 and TIMP-3 overexpression on tube formation by hEMVECs and hFMVECs

Because of the lack of effect of TIMP-1 on tube formation in our previous experiments with VEGF/TNF{alpha}-stimulated hFMVECs (16), we compared the effects of TIMP-1 and TIMP-3 overexpression on capillary-like tube formation by hEMVECs and hFMVECs under identical culture conditions. Both cell types were grown on a fibrin-10% collagen matrix and stimulated by the simultaneous addition of VEGF and TNF{alpha}, which is required to induce tubules by hFMVECs (11). Like in VEGF-stimulated hEMVECs, both TIMP-1 and TIMP-3 reduced capillary-like tube formation in VEGF/TNF{alpha}-stimulated hEMVECs to the same extent as BB94 (Fig. 5Go, striped bars). In contrast, only TIMP-3 inhibited tube formation by hFMVECs to a significant extent. Similar data were obtained with fibrin matrices (Fig. 5Go, black bars). No significant cell detachment was observed in the AdTIMP-1- or AdTIMP-3-transduced hFMVECs and hEMVECs grown on the fibrin matrix, neither under control conditions nor in cells stimulated with VEGF/TNF{alpha} or TNF{alpha} alone (data not shown). This indicates that the overexpression of TIMP-1 or TIMP-3 did not induce a visible degree of apoptosis or cell death under our experimental conditions.



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  		FIG. 5. TIMP-1 inhibits capillary-like tube formation by hEMVECs but not by hFMVECs. hEMVECs and hFMVECs were transduced with 2.5 x 106 pfu/ml AdLacZ, AdTIMP-1, and AdTIMP-3. Subsequently, the cells were cultured on top of a three-dimensional fibrin matrix or a fibrin-10% collagen matrix in M199 supplemented with 10% HS and 10% NBCS and stimulated with VEGF-A (10 ng/ml) and TNF{alpha} (10 ng/ml) with or without BB94 (5 µg/ml). Mean tube length was measured and expressed as a percentage of the tube formation by the AdLacZ-transduced cells ± SEM/range of two to three independent experiments performed in duplicate or triplicate wells (fibrin matrix, black bars; fibrin-collagen matrix, striped bars). The mean tube length of the AdLacZ-transduced hEMVECs was 270 ± 80 mm/cm2 on the fibrin matrix and 266 ± 83 mm/cm2 on the fibrin-collagen matrix. *, P < 0.05 vs. LacZ-transduced cells.

 
Inhibition of MT3-MMP reduces tube formation by hEMVECs

The inhibition of tube formation by both TIMP-1 and TIMP-3 overexpression indicates that MMPs other than MT1-MMP play a role in the regulation of tube formation by hEMVECs. To obtain evidence for the involvement of MT3-MMP in the regulation of this process, tube formation by hEMVECs was induced in the presence of anti-MT3-MMP IgG. Inhibition of MT3-MMP significantly reduced the VEGF-A-enhanced capillary-like tube formation by hEMVECs, whereas nonspecific anti-fluorescein isothiocyanate (anti-FITC) IgG had no effect (Fig. 6Go). The inhibition of VEGF-enhanced tube formation by MT3-MMP IgG was 48.8% of the inhibition achieved by BB94, suggesting that other metalloproteinases may contribute additionally.



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  		FIG. 6. Inhibition of MT3-MMP reduces tube formation by hEMVECs. hEMVECs were cultured on top of a three-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS. Cells were cultured under control conditions in the presence of 0.5 ng/ml VEGF or stimulated with VEGF-A (10 ng/ml), VEGF-A (10 ng/ml) and anti-FITC IgG (25 µg/ml), VEGF-A (10 ng/ml) and anti-MT3-MMP IgG (25 µg/ml), or VEGF-A (10 ng/ml) and BB94 (5 µg/ml). Mean tube length was measured and expressed as the mean tube length ± SEM/range of two experiments performed in duplicate wells. *, P < 0.02 vs. VEGF-stimulated hEMVECs; *, P < 0.05 vs. VEGF/anti-FITC IgG-treated hEMVECs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that both the u-PA/plasmin system and MMPs contribute to the invasion and tubular structure formation by endothelial cells in a three-dimensional fibrin-collagen matrix. Because TIMP-1 and TIMP-3 overexpression reduced capillary-like tube formation by hEMVECs to the same extent, not primarily MT1-MMP, but other MMPs play a regulatory role in this process in hEMVECs. Major MMPs expressed by hEMVECs were MMP-1, MMP-2, MT1-MMP (MMP-14), MT3-MMP (MMP-16), and MT4-MMP (MMP-17) under basal and VEGF-A-stimulated conditions. Our data suggest that MT3-MMP is involved in the regulation of tube formation by hEMVECs, because tube formation by hEMVECs was inhibited of by anti-MT3-MMP IgG in vitro (Fig. 6Go), and MT3-MMP was encountered in endothelial cells of proliferative endometrium in vivo (Fig. 2Go).

Our data on the expression of MMPs by hEMVECs in vitro are in agreement with observations reported from immunohistochemical studies in endometrial tissue sections. Freitas et al. (54) found MMP-1, MMP-2, MMP-3, and MMP-9 in endometrial vascular structures, which might include endothelial cells. MMP-2 was demonstrated in newly formed capillary strands (54). Skinner et al. (55) found MMP-9 on endometrial endothelial cells only after exposure to high progestagen levels. MT1-MMP was detected at low levels on endothelial cells in proliferative and secretory endometrium (36, 37). MT2-MMP was observed at a constant low level throughout the menstrual cycle (35, 36). In addition, TIMP-1, -2, and -3 were demonstrated in endometrial endothelial cells by in situ hybridization (26, 37, 56). Recently, Goffin et al. (35) also reported the presence of MMP-19 mRNA in endometrial tissue throughout the cycle and the mRNAs of MMP-7, MMP-26 and MT3-MMP in this tissue during the proliferative phase of the cycle. However, no information on their expression by specific cells is currently available.

Within the large group of MMPs, the MT-MMPs attract specific attention because of their membrane localization that enables them to regulate localized proteolytic activities directly at the cell-matrix interaction sites. Hotary et al. (28) showed that overexpression of the transmembrane MT1-MMP, MT2-MMP, or MT3-MMP induced endothelial invasion and tube formation in fibrin, whereas the GPI-anchored MT4-MMP was unable to do so. MT1-MMP and MT3-MMP are involved in the migration and invasion of various mesenchymal cells, such as fibroblasts and smooth muscle cells (57), whereas other cells, such as leukocytes and trophoblasts, use MT2-MMP (58, 59). Our data indicate that human endometrial endothelial cells in vitro largely express MT1-MMP, MT3-MMP, and MT4-MMP, whereas only tiny amounts of MT2- and MT5-MMP mRNA are present. Previous studies on human umbilical vein endothelial cells and hFMVECs (14, 15, 16, 28, 53) indicated that invasion and tube formation of endothelial cells was inhibited by TIMP-3 and not by TIMP-1, suggesting that MT1-MMP has a dominant role among the MMPs in regulating endothelial migration and invasion. The present data not only confirm our previous data for hFMVECs but also show consistently that both TIMP-1 and TIMP-3 inhibited tube formation by endometrial endothelial cells. Although these data do not exclude the involvement of MT1-MMP, they strongly suggest that other MMPs than MT1-MMP may contribute more dominantly to endometrial angiogenesis.

The expression of MMP-1 differed markedly between hEMVECs and hFMVECs, although a role for MMP-1 is less likely because MMP-1 is up-regulated only in the secretory phase of the menstrual cycle and not in the proliferative phase. However, data on cell-specific expression are required before definitive conclusions can be drawn. A second possible explanation of the comparable inhibition by TIMP-1 and TIMP-3 might be that MT1-MMP acts in concert with other MMPs, in particular MMP-2, and that inhibition of the other MMPs is rate limiting. However, the comparable expressions of MMP-2 and MT1-MMP in hEMVECs and hFMVECs do not favor this suggestion. Finally, a more likely candidate may be MT3-MMP, which, like MT1-MMP, can contribute potently to angiogenesis in a fibrinous matrix (28). The recent finding that the expression of MT3-MMP mRNA is elevated in endometrial tissue during the proliferative phase of the menstrual cycle suggests such a role (35). Furthermore, our data on the relative expressions of MT3-MMP mRNAs in hEMVECs and hFMVECs, the presence of MT3-MMP protein on endometrial endothelial cells, and the inhibition of capillary tube formation by inhibiting MT3-MMP are strongly in favor of a contribution of MT3-MMP in capillary-like tube formation by hEMVECs.

To summarize, MMPs contribute to in vitro capillary tube formation by human endometrial endothelial cells. Whereas capillary tube formation by hFMVECs depends largely on MT1-MMP, the described data for hEMVECs suggest that other MMPs than MT1-MMP, in particular MT3-MMP, play an important role in tube formation by human endometrial endothelial cells.


   Acknowledgments
 
We thank Dr. R. M. F. van der Weiden (St. Franciscus Gasthuis, Rotterdam, The Netherlands) and Dr. R. A. Verwey and colleagues (Bronovo Hospital, The Hague, The Netherlands), who provided us with endometrial tissue. We thank Dr. R. Kleemann for his advice and help regarding Western blotting.


   Footnotes
 
Part of this study has been sponsored by the Netherlands Heart Foundation, Grant M93-001.

M.P. and K.K. contributed equally to the study.

Abbreviations: bFGF, Basic fibroblast growth factor; CT, number of cycles; dCT, difference in CT values; ECGF, endothelial cell growth factor; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; hEMVEC, human endometrial microvascular endothelial cell; hFMVEC, human foreskin microvascular endothelial cell; hMVEC, human microvascular endothelial cell; HS, human serum; HSA, HS albumin; M199, medium 199; MMP, matrix metalloproteinase; MT, membrane-type; NBCS, newborn calf serum; PMA, phorbol 12-myristate 13-acetate; TIMP, tissue inhibitor of MMP; u-PA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor.

Received May 11, 2004.

Accepted July 21, 2004.


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 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals