Originally published In Press as doi:10.1074/jbc.M201674200 on July 11, 2002
J. Biol. Chem., Vol. 277, Issue 39, 36288-36295, September 27, 2002
Matrix Metalloproteinases Cleave Connective Tissue Growth Factor
and Reactivate Angiogenic Activity of Vascular Endothelial Growth
Factor 165*
Gakuji
Hashimoto
§,
Isao
Inoki,
Yutaka
Fujii¶,
Takanori
Aoki
,
Eiji
Ikeda, and
Yasunori
Okada**
From the Department of Pathology, School of Medicine,
Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-0016, § Research Division, Sumitomo Pharmaceuticals, 3-1-98 Kasugadenaka, Konohana-ku, Osaka, 554-0022, ¶ Department of
Chemistry, Fukui Medical University, 23-3, Shimoaizuki, Matsuoka-cho,
Yoshida-gun, Fukui, 910-1193, and Daiichi Fine Chemical Co.,
Ltd., 530 Chokeiji, Takaoka, Toyama, 933-8511, Japan
Received for publication, February 19, 2002, and in revised form, June 5, 2002
 |
ABSTRACT |
Vascular endothelial growth factor
(VEGF), a potent angiogenic mitogen, plays a crucial role in
angiogenesis under various pathophysiological conditions. We have
recently demonstrated that VEGF165, one of the VEGF
isoforms, binds connective tissue growth factor (CTGF) and that its
angiogenic activity is inhibited in the VEGF165·CTGF
complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H.,
Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and
Okada, Y. (2002) FASEB J. 16, 219-221). In the
present study, we further examined the susceptibility of the
VEGF165·CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of
VEGF165 after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH2-
and COOH-terminal fragments, whereas VEGF165 was completely
resistant to the MMPs. On the other hand, elastase and plasmin cleaved
both CTGF and VEGF165 of the complex, but they were
completely resistant to ADAMTS4. By digestion of the immobilized
VEGF165·CTGF complex with MMP-3 or MMP-7, both
NH2- and COOH-terminal fragments of CTGF were dissociated
and released from the complex into the liquid phase. The in
vitro angiogenic activity of VEGF165 blocked in the
VEGF165·CTGF complex was reactivated to original levels
after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivo
angiogenesis assay using a Matrigel injection model in mice. These
results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF165 suppressed by
the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF
degradation in VEGF-induced angiogenesis during embryonic development,
tissue maintenance, and/or pathological processes of various diseases.
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INTRODUCTION |
Vascular endothelial growth factor (VEGF)1 has been
reported to play a key role in
angiogenic processes under various pathophysiological conditions such
as embryonic development, inflammatory diseases, diabetic retinopathy,
and tumor growth (1-6). Five different splicing variants of VEGF
(VEGF121, VEGF145, VEGF165,
VEGF189, and VEGF206) have been identified so
far (7). The angiogenic activity of VEGF in vivo may be
regulated by the gene expression of the VEGF isoforms and their
receptors, fms-like tyrosine kinase-1 (Flt-1 = VEGFR-1) (8) and
kinase insert domain-containing receptor (KDR = VEGFR-2) (9). However,
another important regulation mechanism is extracellular inhibition of
activity through complex formation with proteins, which include
platelet factor-4 and soluble forms of VEGF receptors (10-12). By
screening a human chondrocyte cDNA library using a yeast two-hybrid
system, we have recently demonstrated that connective tissue growth
factor (CTGF) inhibits VEGF165-induced in vitro
and in vivo angiogenesis through binding to
VEGF165 (13). CTGF is a member of the CCN
(CTGF/cysteine-rich 61/nephroblastoma-overexpressed gene) family and consists
of 349 amino acids with four distinct domains, i.e.
insulin-like growth factor-binding protein (IGFBP), von Willebrand
factor type C repeat (vWFC), thrombospondin type 1 repeat (TSP-1), and
COOH-terminal (CT) domains (14, 15). Because both VEGF and CTGF are
expressed in physiological conditions including growth plate
morphogenesis and wound healing and pathological fibrosis such as
hepatic fibrosis and myocardial fibrosis, the angiogenic activity of
VEGF under pathophysiological conditions may be controlled by
interaction with CTGF. However, this regulation mechanism can be
affected by proteinases, since VEGF and CTGF are susceptible to
proteolytic degradation. Plasmin is known to digest VEGF165
and inactivate its angiogenic activity (16). Several fragments of
CTGF have been detected in culture media of various cells (17),
biological fluids such as sera (17) and uterine luminal flushing (18). However, no information is available for the proteinases that digest
CTGF and modulate the angiogenic activity of the
VEGF165·CTGF complex.
Matrix metalloproteinases (MMPs) are a gene family of structurally and
functionally related zinc endopeptidases that consist of 23 different
members in human (19). MMPs play an essential role in physiological
turnover and pathological degradation of extracellular matrix (ECM)
macromolecules such as proteoglycans and collagens (20). However,
recent studies indicate that MMPs can also digest molecules other than
ECM components. Mcquibban et al. (21) demonstrate that
monocyte chemoattractant protein-3 (MCP-3) is clipped by MMP-2 and that
the cleaved chemokine becomes an antagonist of chemoattractive
activity. Interleukin-1
, Fas ligand, and IGFBP-3 are also
susceptible to MMPs including MMP-1, -2, -3, -7, and/or -9 (22-25).
Thus, it might be possible that MMPs are capable of degrading the
VEGF165·CTGF complex to affect biological activity.
In the present study, we examined the susceptibility of the
VEGF165·CTGF complex to six different MMPs including
MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3
(stromelysin-1), MMP-7 (matrilysin), MMP-9 (gelatinase B), and MMP-13
(collagenase-3) and assayed the angiogenic activity of the complex
after MMP treatment. The data demonstrate for the first time that
MMP-1, -3, -7, and -13 selectively degrade CTGF bound to
VEGF165, which itself is resistant to MMPs, and that the
angiogenic activity of VEGF165 is recovered by dissociation
of CTGF fragments from VEGF165 after digestion.
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EXPERIMENTAL PROCEDURES |
Preparation of Human Recombinant CTGF--
Recombinant CTGF was
prepared as described previously (13). Briefly, CTGF cDNA amplified
by PCR from a chondrocyte cDNA library
(CLONTECH, Palo Alto, CA) was cloned into pCMVtag4a
(Stratagene, La Jolla, CA) and expressed as FLAG-tagged protein.
Culture media were harvested 3 days after transfection of the CTGF
expression vector into COS-7 cells, concentrated by ultrafiltration,
and subjected to anti-FLAG M2-agarose affinity column chromatography (Sigma-Aldrich). Recombinant CTGF was eluted with 6 M urea,
washed with 50 mM Tris-HCl (pH 7.5), 3 M NaCl,
10 mM CaCl2, 0.05% Brij 35 and dialyzed
against 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35 (TNCB buffer)
immediately after elution (13). Purified CTGF showed two bands of 38 and 35 kDa on silver-stained gels after SDS-PAGE, which correspond to
the glycosylated and non-glycosylated forms (13). The biological
activity of recombinant CTGF was confirmed by tube formation assay
(13). Protein concentration was determined using BCA protein assay
reagents (Pierce). Aliquots of purified CTGF were radioiodinated
according to the method of Fraker and Speck (26) and used for binding assay.
Preparation of Human MMPs and Other Proteinases--
The
zymogens of MMP-1, -2, -3, -7, -9, and -13 were purified and activated
by incubation with p-aminophenylmercuric acetate (Sigma-Aldrich) according to previous methods (27-31). Concentrations of MMPs were determined by titration with tissue inhibitors of metalloproteinases as described previously (32, 33). Recombinant human
ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs
4, which is aggrecanase-1) was purified according to our method
(33), and activity was verified in an assay using aggrecan as a
substrate (34). Concentration of ADAMTS4 was determined using BCA
protein assay reagents (Pierce). Plasminogen (Sigma-Aldrich) and
pro-elastase (Calbiochem) were activated with streptokinase (Calbiochem-Novabiochem) and trypsin (Sigma-Aldrich), respectively.
Preparation of Polyclonal Antibodies--
Polyclonal antibodies
against the IGFBP and CT domains of CTGF (anti-IGFBP and anti-CT
antibodies, respectively) were raised in rabbits by injecting bovine
serum albumin (BSA)-conjugated oligopeptides corresponding to the amino
acid sequences of the IGFBP domain (residues 83-95, DFGSPANRKIGVC) and
CT domain (residues 257-273, IRTPKISKPIKFELSGC), respectively. IgG was
isolated from the antisera by DEAE-Sephacel column chromatography.
Specific reaction to CTGF was demonstrated by immunoblotting of culture media of 293T cells (an immortalized human embryonic kidney cell line)
transfected with pCMVtag4a/CTGF plasmid as described previously (13).
Degradation of VEGF165·CTGF Complex by MMPs and
Other Proteinases--
Non-glycosylated recombinant
VEGF165 produced in Escherichia coli was
purchased from Pepro Tech EC Ltd. (London, UK).
VEGF165·CTGF complex was prepared by incubation of
VEGF165 with CTGF at a molar ratio of 1:1 at 4 °C for
24 h as described previously (13). Digestion of the complex was
initially examined by incubation of the substrate at 37 °C for
24 h with MMPs, ADAMTS4, elastase, or plasmin in an
enzyme-to-substrate ratio of 1:30 in TNCB buffer. Because CTGF in the
complex was efficiently digested with MMP-1, -3, -7, and -13, time
course digestion was performed by incubation of the complex with these
MMPs for different periods ranging from 0 to 24 h in the same
enzyme-to-substrate ratio. The reactions were terminated with 20 mM EDTA, and the digestion products were analyzed on
silver-stained gels after SDS-PAGE and by immunoblotting using
anti-IGFBP (1 µg/ml), anti-CT (1 µg/ml), or anti-VEGF antibodies (10 µg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA) according to our method (30). CTGF alone was also digested with MMP-1, -3, -7, and -13 at 37 °C for 24 h to confirm the digestion of CTGF
without complex formation.
NH2-terminal Sequence Analyses--
CTGF (10 µg)
was incubated with MMP-1, -3, -7 or -13 at an enzyme-to-substrate ratio
of 1:30 at 37 °C for 24 h for MMP-1, -3, and -7 and for 4 h for MMP-13 in a total reaction volume of 100 µl in TNCB buffer.
Reactions were stopped by treatment with 20 mM EDTA, and
the digestion products were subjected to SDS-PAGE. Proteins in the gels
were transferred to polyvinylidene difluoride membranes and located by
staining with 0.1% Coomassie Brilliant Blue R-250. The bands of
interest were excised and sequenced by Edman degradation using Procise
491 Protein Sequencer (PerkinElmer Life Sciences) (33).
Release of CTGF Fragments from the VEGF165·CTGF
Complex after Digestion with MMPs--
Microtiter plates with 96 wells
(Immunomodule, NalgeNunc, Rochester, NY) were coated with 50 µl of
VEGF165 (500 ng/well) for 16 h at 4 °C. Then the
plates were washed twice with phosphate-buffered saline (PBS)
containing 0.05% Brij 35 and subsequently blocked with 1% BSA in PBS
for 2 h at room temperature. 125I-Labeled CTGF
(1~3 × 105 cpm, 10 ng/well) was bound to each well
by incubation for 24 h at 4 °C. After washing carefully twice
with PBS containing 0.05% Brij 35, the bound proteins were digested
with MMP-3 (1 ng) or MMP-7 (1 ng) in TNCB buffer or buffer alone for
0.5, 4, or 24 h at 37 °C. Reaction mixtures were collected, and
CTGF fragments were immunoprecipitated with 50 µl of anti-IGFBP
antibody or anti-CT antibody, which were then trapped onto protein
G-Sepharose beads (Amersham Biosciences). CTGF remaining on the wells
was dissociated by treatment with 1 N NaOH. Radioactivity
of the precipitates and NaOH-dissociated fractions was counted by
-counter ARC-600 (Aloka, Tokyo, Japan).
Endothelial Cell Tube Formation Assay--
Tube formation assay
using bovine aortic endothelial cells (BAEC) in type I collagen gel was
carried out as described previously (13). Briefly, type I collagen (3 mg/ml) (Vitrogen, Cohesion, Palo Alto, CA) was mixed with 10-fold
concentrated M199 (Invitrogen) and 0.1 N NaOH at a ratio of
8:1:1, respectively. The mixture (500 µl) was dispensed to 24-well
culture plates and incubated at 37 °C for 1 h to form collagen
fibrils. BAEC were trypsinized and plated on the collagen gel at a
density of 5 × 104 cells/well. They were cultured in
M199 medium supplemented with 10% fetal bovine serum overnight. After
removal of the media, 500 µl of the collagen mixture was overlaid on
the cells and incubated for 1 h at 37 °C. The cells were
stimulated for 3 days with VEGF165, CTGF,
VEGF165·CTGF complex, VEGF165·CTGF complex
digested with MMPs or MMPs by adding media containing these molecules
onto the top of the collagen gel layers. For VEGF165·CTGF
complex formation, VEGF165 (40 ng) was incubated for
24 h at 4 °C with increasing concentrations of CTGF (40 and 200 ng and 1 µg) in M199 medium containing 1% fetal bovine serum.
Digestion mixtures of the complex, which were made by incubation of
VEGF165 (40 ng) with CTGF (100 ng) for 24 h at
4 °C, were prepared by digestion of the complex with MMP-1, -3, and
-13 (3 ng each) for 24 h at 37 °C. To demonstrate that
recovered angiogenic activity after digestion of the
VEGF165·CTGF complex with MMP-1, -3, or -13 is derived
from VEGF165, the MMP digestion mixtures prepared as
mentioned above were incubated with 40 µg of mouse monoclonal
anti-VEGF IgG (Upstate Biotechnology, Lake Placid, NY) or non-immune
mouse IgG (DAKO, Glostrup, Denmark) for 1 h at 37 °C and then
used for tube formation assay. As for a control, the cells were also
stimulated with CTGF (100 ng) or CTGF (100 ng) digested with MMP-1, -3, or -13 (3 ng each) for 24 h at 37 °C. After 3 days of culture,
each well was photographed, and the tubular length of the cells was
measured in five different areas in 0.25 mm2 using NIH
Image 1.62 as described previously (13). The length was expressed as
mm/mm2. Experiments were repeated three times, and similar
results were obtained.
In Vivo Angiogenesis Assay--
In vivo angiogenesis
assay using a Matrigel injection model was performed as previously
described (13). Before injection, 500 µl of Matrigel (Collaborative
Biomedical Products, Bedford, MA) was mixed with 50 µl of PBS,
VEGF165 (50 ng), VEGF165 (50 ng)-CTGF (100 ng)
complex, complex digested with MMP-3 (3 ng) for 24 h at 37 °C,
MMP-3 digestion mixture incubated with anti-VEGF IgG (50 µg; Upstate
Biotechnology) or non-immune mouse IgG (50 µg; DAKO) for 1 h at
37 °C, CTGF (100 ng), CTGF (100 ng) digested with MMP-3 (3 ng) for
24 h at 37 °C, or MMP-3 (3 ng). MMP-3 activity was inactivated
before mixing with Matrigel by incubation with sheep polyclonal
anti-human MMP-3 IgG (0.5 µg) (35). Matrigel containing these factors
was injected subcutaneously near the abdominal midline of 4-week-old
male C57BL/6J mice. Cutaneous tissues with Matrigel plugs were removed
5 days after injection, fixed in periodate-lysine-paraformaldehyde, and
embedded in paraffin. The serial sections were stained with hematoxylin
and eosin and immunostained with rabbit anti-vWF antibody (1:200; DAKO)
or non-immune rabbit IgG. The degree of angiogenesis was determined by
counting vWF-positive cells and blood vessels with an apparent luminal area/mm2 area using NIH Image 1.62 according to our
previous method (13).
Statistical Analysis--
Measured values were expressed as
mean ± S.D. In the tube formation assay, the difference of
tubular length treated with VEGF165 and
VEGF165·CTGF complex was analyzed by Bonferroni/Dunn
test. In the in vivo angiogenesis assay, the differences of
spindle cells and blood vessels between two independent groups were
analyzed by the Mann-Whitney test. These tests were performed using
StatView 5.0. p values of less than 0.05 were considered significant.
| |
RESULTS |
Inhibition of Angiogenic Activity of VEGF165 by
CTGF--
Our previous studies (13) have demonstrated that the
angiogenic activity of glycosylated VEGF165 is inhibited
with CTGF by in vitro and in vivo assays. To
further confirm the inhibitory effect of CTGF on the angiogenic
activity of non-glycosylated recombinant VEGF165 used in
the present study, the activity was assayed by tube formation assay. As
shown in Fig. 1, A and
B, VEGF165 stimulated the tubular extension
5.5-fold more than the untreated control. In contrast,
VEGF165-induced tube formation was remarkably reduced in
the presence of CTGF. Increasing concentrations of CTGF inhibited the
activity with a peak at 200 ng/ml. These data are consistent with our
previous data of CTGF inhibition of angiogenesis using glycosylated
VEGF165 (13), indicating that glycosylated chains of
VEGF165 are not involved in the inhibitory effect of
CTGF.
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Fig. 1.
Effect of CTGF on VEGF165-induced
tube formation. A, representative micrographs of
VEGF165-induced tube formation of BAEC in the presence of
CTGF. BAEC were stimulated for 3 days with 40 ng/ml VEGF165
in the presence of 0 (2), 40 (3), 200 (4), or 1000 ng/ml CTGF (5) and photographed.
Panels 1 and 6 are the controls cultured with
medium and 1000 ng/ml CTGF alone, respectively. B, the
effect of CTGF on VEGF165-induced tube formation. Tubular
length of the cells was measured in 5 different areas of 0.25 mm2 using NIH Image, and the length was expressed as
mm/mm2 as described under "Experimental Procedures."
Bars, S.D. **, p < 0.01 as compared with
VEGF treatment.
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Digestion of VEGF165·CTGF Complex by MMPs or Other
Proteinases--
VEGF165 and CTGF were complexed by
incubating them at a molar ratio of 1:1 for 16 h at 4 °C, and
then the complex was digested at 37 °C for 24 h with 6 different MMPs (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4, elastase, or
plasmin. When the reaction products of the VEGF165·CTGF
complex with MMPs were analyzed on silver-stained gels, CTGF of 35 and
38 kDa was cleaved by MMP-1, -3, -7, and -13 into a few fragments with
lower molecular weights ranging from 19 to 23 kDa (data not shown).
However, because VEGF165 of 19 kDa co-migrated with these
fragments, some digestion fragments could not be differentiated from
VEGF165. On the other hand, when the reaction mixtures were
analyzed by immunoblotting with two different anti-CTGF antibodies
specific to the IGFBP domain or CT domain, intact CTGF disappeared, and
several 19~23-kDa bands immunoreactive with these antibodies were
observed in the digestion products with MMP-1, -3, -7, and -13 (Fig.
2, A and B). In
addition, weak immunoreactive bands were also detected by digestion
with MMP-2 and MMP-9 (Fig. 2, A and B), although
they were not detectable on silver-stained gels (data not shown). CTGF
was also digested by elastase and plasmin into small peptides, most of
which were not observed by immunoblotting with antibodies (Fig. 2,
A and B), or on a silver-stained gel (data not
shown). However, CTGF was resistant to ADAMTS4 (Fig. 2, A
and B). In contrast to the susceptibility of CTGF to MMPs,
VEGF165 was completely resistant to digestion with MMPs and
ADAMTS4 under these conditions, whereas both elastase and plasmin
cleaved VEGF165 of 19 kDa into 13-kDa fragments (Fig.
2C).
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Fig. 2.
Immunoblotting analyses of the digestion
products of VEGF165·CTGF complex after incubation with
MMP-1, -2, -3, -7, -9, -13, ADAMTS4, elastase, or plasmin. The
VEGF165·CTGF complex was digested with MMP-1 (lane
2), MMP-2 (lane 3), MMP-3 (lane 4), MMP-7
(lane 5), MMP-9 (lane 6), MMP-13 (lane
7), ADAMTS4 (lane 8), elastase (lane 9), or
plasmin (lane 10) for 24 h at 37 °C as described
under "Experimental Procedures." After termination of the reaction,
the digestion products were subjected to SDS-PAGE (15% total
acrylamide) under reduction and analyzed by immunoblotting using
anti-IGFBP (A), anti-CT (B), and anti-VEGF
antibodies (C). Lane 1 is the control incubated
with buffer alone for 24 h at 37 °C.
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Time Course Digestion of VEGF165·CTGF Complex by
MMP-1, -3, -7, and -13--
To compare the susceptibility of the
VEGF165·CTGF complex to degradation with MMP-1, -3, -7, and -13, all of which digested CTGF in a 24-h incubation, time course
digestion was carried out and monitored by immunoblotting with anti-CT
antibody. Among the four MMPs examined, MMP-13 appeared to most
efficiently digest CTGF in the complex, since intact CTGF was degraded
fastest. A complete digestion of CTGF was observed after a 4-h
incubation with MMP-13 (Fig.
3D) and after an 8-h
incubation with MMP-3 (Fig. 3B). However, MMP-1 and MMP-7
required a 24-h incubation (Fig. 3, A and C).
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Fig. 3.
Time course digestion of the
VEGF165·CTGF complex with MMP-1, -3, -7, and -13. Digestion of the VEGF165·CTGF complex was performed by
incubation with MMP-1 (A), -3 (B), -7 (C), or -13 (D) at 37 °C for 0-24 h as
described under "Experimental Procedures." After termination of the
reaction, the digestion products were analyzed by immunoblotting using
anti-CT antibody.
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Determination of CTGF Cleavage Sites by MMPs--
When CTGF alone
was digested with MMP-1, -3, -7, and -13 and the digestion patterns of
CTGF were compared with those obtained by VEGF165·CTGF
complex digestion with MMPs, the fragments detected with anti-IGFBP and
anti-CT antibodies were identical between the samples of CTGF alone and
VEGF165·CTGF complex (data not shown). Thus,
NH2-terminal sequence analyses of the CTGF fragments
generated by digestion with MMP-1, -3, -7, and -13 were performed by
incubation of CTGF alone with MMPs. As shown in Table
I, the NH2-terminal sequences
of the COOH-terminal fragments of CTGF were successfully determined.
However, the NH2-terminal sequence of intact CTGF was not
obtained, indicating that its NH2 terminus is blocked. Besides intact CTGF, the NH2-terminal sequences of several
19~23-kDa CTGF fragments recognized with anti-IGFBP antibody could
not be obtained, suggesting that these fragments contain the
NH2 terminus of CTGF. By CTGF digestion with these MMPs,
CTGF was cleaved at several peptide bonds including the
Ala181-Tyr182 (MMP-3 and -7),
Arg183-Leu184 (MMP-7 and -13),
Met194-Ile195 (MMP-1, -3, -7, and -13), or
Cys199-Leu200 bond (MMP-1, -7, and -13) (Table
I), all of which are located between the vWFC and TSP domains. Among
them, the Met194-Ile195 bond was considered to
be the common site susceptible to digestion with all these MMPs.
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Table I
NH2-terminal sequences of the COOH-terminal fragments generated
by digestion with MMP-1, -3, -7, and -13. The NH2-terminal
sequences in parentheses are those of minor components.
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Dissociation and Release of CTGF Fragments from the
VEGF165·CTGF Complex by Digestion with MMPs--
When
125I-CTGF was incubated in wells coated with
VEGF165 or BSA, a large amount of 125I-CTGF
specifically bound to VEGF165-coated wells but not to those BSA-coated (Fig. 4A). The
radioactivity of 125I-CTGF bound to VEGF165 on
the wells decreased with time of MMP-3 digestion and reached almost
background levels after 4- and 24-h incubations (Fig. 4B),
suggesting dissociation and release of the CTGF digestion fragments
into the supernatant. In accordance with our prediction,
immunoprecipitation of the NH2- and COOH-terminal 125I-CTGF fragments with anti-IGFBP and anti-CT antibodies
demonstrated that radioactivity of fragments increases in the liquid
phase, and the sum of the radioactivity is nearly equal to that bound to VEGF165 after a 24-h incubation (Fig. 4B).
Similar results were obtained in digestion experiments using MMP-7
(data not shown). By buffer incubation of the complex on the wells in
the absence of MMP-3 or MMP-7, release of the radioactivity was only
negligible at each incubation time (data not shown). Thus, these data
indicate that both MMPs digest CTGF and dissociate its fragments from
the VEGF165·CTGF complex.
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Fig. 4.
Release of CTGF fragments from the
VEGF165·CTGF complex after the digestion with MMP-3.
A, the specific binding of 125I-CTGF to
VEGF165 immobilized on microtiter plates. Binding activity
of 125I-CTGF was assayed by using microtiter plates coated
with BSA or VEGF165 as described under "Experimental
Procedures." B, dissociation and release of the CTGF
fragments from the VEGF165·CTGF complex after digestion
with MMP-3. VEGF165·125I-CTGF complex was
formed on microtiter plates and digested with MMP-3 for 0.5, 4, and
24 h at 37 °C as described in under "Experimental
Procedures." The NH2-terminal and COOH-terminal digestion
fragments of 125I-CTGF were immunoprecipitated with
anti-IGFBP and anti-CT antibodies, respectively. The radioactivity of
125I-CTGF bound to VEGF165-coated wells
(closed column), NH2-terminal CTGF fragments
(shaded column), and COOH-terminal fragments (hatched
column) were measured by -counter. Bars, S.D. of
triplicate assays.
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Recovery of Tube Formation Activity of the
VEGF165·CTGF Complex after Digestion with
MMPs--
Angiogenic activity of the VEGF165·CTGF
complex treated with MMP-1, -3, and -13 was assayed by tube formation
assay. VEGF165-mediated stimulation of endothelial tubular
extension was significantly inhibited by complex formation with CTGF
(Fig. 5, A, 1-3,
and B). However, when the complex was digested with MMP-1,
-3, or -13 and then the digestion products were applied to the tube
formation assay, angiogenic activity recovered to the original levels
obtained by VEGF165 treatment (Fig. 5, A,
4-6, and B). The MMPs themselves showed no definite effects on tube formation (Fig. 5, A,
7-9, and B). CTGF alone tended to
have weak angiogenic activity, although the activity was not
significantly different from the buffer control (Fig. 5C).
However, CTGF fragments prepared by the digestion of CTGF alone with
MMP-1, -3, or -13 exhibited no definite angiogenic activity (see Fig.
5C for digestion with MMP-3; data not shown for digestion
with MMP-1 or MMP-13). To further demonstrate that VEGF165
is responsible for the reactivated angiogenic activity in the
MMP-cleaved VEGF165·CTGF complex, the effect of anti-VEGF IgG on the tube formation activity was examined. As shown in Fig. 5D, the recovered angiogenic activity in the MMP-3-digested
VEGF165·CTGF complex was significantly inhibited to the
level of the VEGF165·CTGF complex, whereas non-immune IgG
treatment had no inhibitory effect.
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Fig. 5.
Recovery of the VEGF165-induced
angiogenic activity in vitro after digestion of the
VEGF165·CTGF complex with MMP-1, -3, and -13. A, representative micrographs of tube formation of BAEC
treated with VEGF165, VEGF165·CTGF complex,
and the complex digested with MMP-1, -3, or -13. BAEC were cultured for
3 days with PBS (1), VEGF165 (2),
VEGF165·CTGF complex (3), or the complex
digested with MMP-1 (4), MMP-3 (5), or MMP-13
(6) and photographed as described in under "Experimental
Procedures." As for controls, the cells were treated with MMP-1
(7), MMP-3 (8), or MMP-13 alone (9).
B and C, effects of MMP-1, -3, or -13 digestion
of the VEGF165·CTGF complex (B) and MMP-3
digestion of CTGF alone (C) on tube formation. Tubular
length of BAEC was measured in 5 different areas of 0.25 mm2 using NIH Image, and the length was expressed as
mm/mm2, as described under "Experimental Procedures."
D, effect of anti-VEGF IgG on the tube formation activity in
the MMP-3-digested VEGF165·CTGF complex. VEGF/CTGF
indicates the VEGF165·CTGF complex. Tubular length was
measured as described above. As for a control, non-immune mouse IgG
(N.I.) was used. Bars, S.D. *, p < 0.05; **, p < 0.01.
|
|
Recovery of in Vivo Angiogenic Activity of the
VEGF165·CTGF Complex after Digestion with MMP-3--
To
further study the effect of MMP digestion of the
VEGF165·CTGF complex on in vivo angiogenesis,
a Matrigel injection model was used. VEGF165 in Matrigel
remarkably stimulated migration of the spindle-shaped cells and blood
vessels with lumina as compared with control without growth factor
(Fig. 6A, 1 and
2). VEGF165-induced blood vessels often reached
the middle of the plugs and appeared to be dilated. Most of the
spindle-shaped cells and the vessel-forming cells within the plugs were
strongly immunostained with anti-vWF antibody but negative with
non-immune rabbit serum (Fig. 6A, 8 and
9). The numbers of the vWF-positive spindle-shaped cells and blood vessels in Matrigel were significantly higher by 6.4-fold in
samples with VEGF165 (300 ± 50 cells/mm2
and 122 ± 16 vessels/mm2) than in those of control
(47 ± 7 cells/mm2 and 19 ± 6 vessels/mm2) (Fig. 6, B and C). CTGF
significantly inhibited the angiogenic activity of VEGF165
to 28 and 47% of the original activity (84 ± 18 cells/mm2 and 57 ± 9 vessels/mm2) (Fig.
6, A (3), B, and C),
although CTGF itself showed weak effects on in vivo
angiogenesis (63 ± 11 cells/mm2 and 29 ± 5 vessels/mm2) (Fig. 6, A (5),
B, and C). More importantly, the digestion
product of the VEGF165·CTGF complex with MMP-3, but not
the CTGF fragments prepared by digestion of CTGF alone with MMP-3,
significantly increased 3.7- and 2.4-fold the angiogenic activity
(314 ± 64 cells/mm2 and 136 ± 36 vessels/mm2) as compared with that of the
VEGF165·CTGF complex (Fig. 6, A (4,
8, and 9), B, and C). This
reactivation of the angiogenic activity in the MMP-3-digested
VEGF165·CTGF complex was inhibited to the level of the
complex by the treatment with anti-VEGF IgG (110 ± 18 cells/mm2 and 72 ± 10 vessels/mm2) (Fig.
6, A (6), B, and C). MMP-3 inactivated
with polyclonal anti-MMP-3 IgG showed no effects on in vivo
angiogenesis (50 ± 6 cells/mm2 and 23 ± 4 vessels/mm2) (Fig. 6, A (7),
B, and C).
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 6.
Recovery of the VEGF165-induced
angiogenic activity in vivo after digestion of the
VEGF165·CTGF complex with MMP-3. A,
representative micrographs of the in vivo angiogenesis using
a Matrigel injection model. Matrigel containing PBS (1),
VEGF165 (2), VEGF165 complexed with
CTGF (3), VEGF165·CTGF complex digested with
MMP-3 (4, 8, and 9), CTGF alone
(5), MMP-3-digested VEGF165·CTGF complex
treated with anti-VEGF antibody (6), or MMP-3 alone
(7) was injected subcutaneously into C57BL/6J mice, as
described under "Experimental Procedures." For each aliquot
digested with MMP-3, activity was blocked by incubation with anti-MMP-3
IgG before mixing with Matrigel. Cutaneous tissues with Matrigel plugs
were removed 5 days after injection, fixed, and embedded in paraffin.
The sections were stained with hematoxylin and eosin (1-7) or
immunostained with anti-vWF antibody (8) or non-immune rabbit serum
(9). Bar, 50 µm for 1-7 and 100 µm for
8 and 9. B and C,
evaluation of the degree of angiogenesis by counting the number of
vWF-positive spindle cells (B) and blood vessels
(C) in the Matrigel plugs with PBS, VEGF165,
VEGF165·CTGF complex, MMP-3-digested complex in the
absence or presence of anti-VEGF IgG, CTGF alone, MMP-3-digested
CTGF, or MMP-3 alone. The bars represent the mean ± S.D. of six mice. **, p < 0.01.
|
|
| |
DISCUSSION |
In the present study, we have demonstrated for the first time that
all six different MMPs, i.e. MMP-1, -2, -3, -7, -9, and -13, can selectively degrade CTGF in the VEGF165·CTGF complex, although the proteolytic activity of MMP-2 and MMP-9 to CTGF was very
weak. Because these MMPs produced identical digestion fragments by
incubation with CTGF alone, the degradation can be ascribed to the
specific recognition of CTGF substrate and its subsequent proteolysis
by MMPs. The primary substrates of MMPs are thought to be ECM
macromolecules, but this study indicates that growth factor is also a
substrate of many MMPs. Actually, accumulated lines of evidence have
indicated that MMPs can cleave various molecules other than ECM
components such as MCP-3, interleukin-1, Fas ligand, and IGFBP-3
(21-24). Our data on the NH2-terminal sequences of the
CTGF fragments generated by digestion with MMP-1, -3, -7, and -13 demonstrate that the residues in the P1' position are Leu, Ile, and
Tyr, all of which are the common residues found in the P1' position for
the ECM and non-ECM substrates of MMPs (20-22,36). In contrast to the
MMPs, ADAMTS4, which cleaves almost selectively the Glu-X bonds of
aggrecan (37) and brevican (33), did not attack CTGF. Among the members
of the MMP gene family, MMP-7 most preferentially degrades ECM
substrates (38). However, this was not the case with CTGF digestion,
since time course digestion showed that MMP-7 appears to have weaker
activity than MMP-13 and MMP-3. The reason is not clear in the present
study. However, because one of the most prominent structural
differences of MMP-7 from other MMPs is the lack of the COOH-terminal
hemopexin-like domain, that plays a key role in the binding to their
substrates (39), the COOH-terminal domains of MMP-13 and MMP-3, in
addition to their catalytic domains, may be required for efficient
digestion of CTGF.
Although CTGF was susceptible to the MMPs examined, cleavage was
limited to the bonds located in the linker region between the vWFC and
TSP-1 domains. Thus, the digestion resulted in the formation of the
major NH2- and COOH-terminal fragments of ~20 kDa. This
contrasts to the digestion patterns with leukocyte elastase and
plasmin, since they degraded CTGF into small peptides by cleaving at
multiple sites after a 24-h incubation. Previous immunoprecipitation studies (17) show that human biological fluids such as sera, cerebrospinal fluids, and ascites contain CTGF degradation fragments of
32, 24, and 18 kDa as well as intact 38-kDa CTGF. These fragments are
also detected in the culture media from human breast cancer cells (17).
Because the molecular sizes of the latter two fragments are similar to
those found after digestion with MMPs and such biological fluids and
culture media usually contain MMPs, it might be possible to speculate
that the 24- and 18-kDa fragments are generated by digestion of CTGF
with MMPs. The COOH-terminal fragments of 10 kDa, which stimulate DNA
synthesis in fibroblasts and smooth muscle cells (18), have been
purified from pig uterine luminal flushing (18) and culture media of
mouse and human fibroblasts (40). In the present study, however, such
fragments were not identified by digestion with MMPs, leukocyte
elastase, or plasmin. Thus, another proteinase(s) than the MMPs and the
neutrophil serine proteinases may be involved in the formation of the
bioactive 10-kDa fragments.
VEGF165 is known to be susceptible to degradation by
plasmin, trypsin, chymotrypsin, clostripain, and bromelain (16). The present study confirmed the data of VEGF165 digestion with
plasmin but further showed that it is also cleaved by leukocyte
elastase. Digestion with these serine proteinases produced a major
VEGF165 fragment with a molecular mass ~6 kDa lower than
recombinant VEGF165 under reducing conditions. Although the
present study did not determine the NH2-terminal sequence
of the fragment, the molecular size suggests that the fragment is
generated through the cleavage at the
Arg110-Ala111 bond of VEGF165 by
plasmin as reported by Keyt et al. (16). In contrast to the
effect of the serine proteinases, our study demonstrated that
VEGF165 is completely resistant to all the six MMPs
examined. These results are in accordance with the finding that serine
proteinase activity but not metalloproteinase activity in the wound
fluid from chronic ulcers is responsible for the degradation of
VEGF165 (41).
One of the most important findings in the present study is that
angiogenic activity of VEGF165, which was suppressed by the complex formation with CTGF, recovered after digestion of CTGF in the
VEGF165·CTGF complex with MMPs. Our previous study
demonstrated that CTGF inhibits VEGF165-induced
angiogenesis by interrupting the binding of VEGF165 to its
major receptor VEGFR-2 through complex formation between the exon
7-coded region of VEGF165 and the TSP-1 domain of CTGF
(13). Thus, the dissociation of CTGF fragments from the
VEGF165·CTGF complex after MMP digestion is essential to
the recovery of angiogenic activity. In the present study, we have
demonstrated that the NH2- and COOH-terminal fragments of
CTGF are dissociated and released from the complex by
digestion with MMP-3 and MMP-7. Because these MMPs cleave the linker
region of CTGF between the vWFC and TSP-1 domains without attacking the binding site (the TSP-1 domain), conformational changes of CTGF caused
by digestion appear to be responsible for dissociation. Our previous
and present studies showed that CTGF itself is an angiogenic factor.
However, the maximal activity of CTGF was weak and confined to only
~25 and ~35% that of VEGF165 in in vitro tube formation assay and in in vivo Matrigel injection
assay, respectively (13). In addition, our data in the present study could demonstrate that the CTGF digestion fragments by MMPs have no
angiogenic activity in in vitro and in vivo
angiogenesis assays. The data are accordant with the previous finding
that the bioactive 10-kDa COOH-terminal CTGF fragment cannot stimulate
endothelial cells (18). Furthermore, angiogenic activity recovered from the VEGF165·CTGF complex after digestion with MMPs was
blocked with anti-VEGF antibody. It is, therefore, concluded that
reactivation of the angiogenic activity in the MMP-digested
VEGF165·CTGF complex is ascribed to the effect of
VEGF165 dissociated from the complex on endothelial cells.
Because of the overexpression of CTGF in tissues undergoing fibrosis
and its stimulatory activity toward fibroblasts to proliferate and
synthesize ECM, CTGF is believed to play a key role in pathological fibrosis such as hepatic fibrosis, atherosclerosis, and myocardial fibrosis (42-44). CTGF is also expressed in the processes of wound healing (41, 45), where granulation with neovascularization precedes
the final scar formation. Interestingly, MMPs are overproduced in the
active angiogenic stages in these pathophysiological conditions (46).
Thus, we hypothesize that in the angiogenic stage of these conditions,
activity of the VEGF165·CTGF complex is in favor of VEGF165 by degradation of CTGF with MMPs, and then in the
fibrotic stage, angiogenic activity is blocked by complex formation
with CTGF because of decreased MMP activity. In cartilage, VEGF, CTGF, and MMPs including MMP-3 are produced in the hypertrophic chondrocytes of the growth plate (47-50). Because the activity of VEGF is essential to the remodeling of the growth plate (47), a similar regulation mechanism of the VEGF·CTGF complex by MMPs might be involved in this
process. A previous study on MMP-9-deficient mice indicated that
development of the growth plate is transiently retarded due to impaired
angiogenesis (51). Because MMP-9 weakly digests CTGF in the
VEGF165·CTGF complex, it might be possible to speculate that this transient phenotype is caused by decreases in the CTGF degradation and subsequent VEGF release from the complex by an action
of MMP-9 in the early developmental stages of the mice before other
MMPs compensate for the lack of MMP-9. Articular cartilage is an
avascular tissue, but diseased cartilage is invaded by pannus tissue,
which is a granulation tissue with hypervascularity (52). VEGF, CTGF,
and MMPs are all expressed in the articular cartilages from patients
with osteoarthritis or rheumatoid arthritis (20,
53).2 Thus, angiogenic
activity of VEGF, which is probably inhibited by VEGF·CTGF complex
formation in normal articular cartilage, may be reactivated by CTGF
degradation with MMPs overproduced in such joints, allowing the pannus
tissue to invade the cartilage. These hypotheses on the in
vivo proteolytic regulation of the VEGF·CTGF complex by MMPs
remain to be further studied at the cellular and tissue levels.
| |
ACKNOWLEDGEMENTS |
We thank Michiko Uchiyama for technical
assistance, Dr. Takayuki Shiomi and Dr. Hiroyuki Nakamura for helpful
advice, and Drs. Takashi Katsumata, Hiroshi Takada, and Yoshihiko Koga
of Sumitomo Pharmaceuticals for encouragement. We are also grateful to
Dr. Atsushi Suzuki for critical reading our manuscript.
| |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence and reprint requests should be addressed.
Tel.: 81-3-5363-3763; Fax: 81-3-3353-3290; E-mail:
okada@sc.itc.keio.ac.jp.
Published, JBC Papers in Press, July 11, 2002, DOI 10.1074/jbc.M201674200
2
G. Hashimoto and Y. Okada, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
ADAMTS, a disintegrin and metalloproteinase
with thrombospondin motifs;
BAEC, bovine aortic endothelial cells;
BSA, bovine serum albumin;
CT, COOH-terminal;
CTGF, connective tissue growth
factor;
ECM, extracellular matrix;
Flt-1, fms-like tyrosine
kinase-1;
IGFBP, insulin-like growth factor binding protein;
MCP-3, monocyte chemoattractant protein-3;
MMP, matrix metalloproteinase;
PBS, phosphate-buffered saline;
TSP-1, thrombospondin type 1 repeat;
vWFC, von Willebrand (vWF) factor type C repeat.
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