(Received for publication, March 13, 1995; and in revised form, July 31, 1995)
From the
Angiogenesis requires degradation of vascular basement membrane
prior to migration and proliferation of endothelial cells; proteinases
are essential ingredients in this process. Because of thrombin's
multiple effects on endothelium, we have examined its role in matrix
metalloproteinase activation using human umbilical vein endothelial
cells. Gelatin zymography of endothelial conditioned media revealed a
prominent 72-kDa progelatinase A band. Addition of -thrombin to
endothelial cells resulted in the generation of 64 and 62 kDa
gelatinolytic bands which is consistent with the activation of
progelatinase A; thrombin had no effect in the absence of cells. This
effect requires the proteolytic site of thrombin since progelatinase A
activation was abolished by specific inhibitors of thrombin. Matrix
metalloproteinase inhibitors diminished thrombin-induced activation of
progelatinase A. Pretreatment of endothelial cells with excess tissue
inhibitor of metalloproteinase-2 or a COOH-terminal fragment of
progelatinase A abrogated thrombin-mediated activation of progelatinase
A presumably by competing with the COOH terminus of native
progelatinase A for interaction with an activator site on endothelial
plasma membranes. Although membrane-type matrix metalloproteinase was
demonstrated in endothelial cells by Northern and Western blotting, the
receptor function of this molecule in thrombin-induced activation of
progelatinase A needs to be clarified. Progelatinase A activation did
not require intracellular signal transduction events mediated by the
thrombin receptor. These data demonstrate that 1) endothelial cells
express a novel activation mechanism for progelatinase A, 2)
proteolytically active thrombin regulates this activation mechanism,
and 3) activation occurs independently of the functional thrombin
receptor.
Whereas the effect of thrombin (EC 3.4.21.5) on production of fibrin and activation of platelets has been intensively studied over many years, interest in the role of thrombin on endothelial function has lagged. Recent studies have indicated that thrombin affects post-clotting events involved in angiogenesis(1) . The vascular endothelium actively binds coagulation proteins, resulting in cell-surface generation of thrombin that can persist within the protected environment of a clot(2) . A unique G-protein-coupled thrombin receptor (3) is known to be functionally expressed by endothelial cells(4) . Interaction of thrombin with an endothelial cell-surface thrombin receptor(s) results in a multiplicity of effects including cell retraction and permeability, generation of phosphoinositides and prostaglandin, and secretion of Von Willebrand factor, tissue plasminogen activator, and platelet-derived growth factor(5, 6, 7) . The role of individual thrombin receptors and endothelial signal transduction events on thrombin-mediated cell activation phenomena remain incompletely characterized(4) .
Neoangiogenesis, the formation of new
blood vessels from preexisting vessels, requires the degradation of
underlying basement membranes prior to migration and proliferation of
endothelial cells, and ultimately the formation of new capillary
sprouts(8) . Matrix metalloproteinases, specifically gelatinase
A (72-kDa type IV collagenase, MMP()-2, EC 3.4.24.24),
gelatinase B (92-kDa type IV collagenase, MMP-9, EC 3.4.24.35), and
interstitial collagenase (MMP-1, EC 3.4.24.7), are released by
endothelial cells and play an important role in turnover of basement
membrane type IV collagen, type I collagen, laminin, and fibronectin
during angiogenesis(9, 10) . A delicately orchestrated
balance between production, activation, and inhibition of MMPs is
considered to be essential in maintenance of blood vessel
integrity(11, 12, 13) . A similar role in
angiogenesis has been proposed for plasminogen activators and
inhibitors with evidence that these two proteolytic systems may be
interconnected(11, 14) .
The physiologic mechanism
controlling the activation of MMPs, especially progelatinase A, is not
well understood(15) . Plasmin and stromelysin are capable of
activating latent gelatinase B and interstitial collagenase, but the
alignment of propeptides in progelatinase A lacks the appropriate
proteinase cleavage sites in the amino-terminal domain of the molecule
required for enzyme activation (16) . Another mechanism
recently described with lectin-stimulated fibroblasts and tumor cells
involves plasma membrane activation of progelatinase A by a contact
requiring
mechanism(17, 18, 19, 20, 21) .
Endothelial (22) and tumor plasma membrane-bound
metalloproteinases (23) have been previously demonstrated to
concentrate proteinases at points of cell surface contact with the
matrix(24) , thus facilitating local tissue degradation. It has
been proposed that the COOH-terminal domain of progelatinase A binds to
a cell surface membrane component (25) leading to the
activation of progelatinase A (17, 18) possibly by
causing a conformational change in progelatinase A that leads to
autoproteolytic processing of the amino-terminal domain(20) .
Recently, Sato et al.(26) have reported the isolation
and cloning of a complementary DNA encoding a previously unidentified
63-kDa matrix metalloproteinase with a transmembrane domain (MT-MMP).
Expression of the gene product on the cell surface induces activation
of progelatinase A in vitro and enhanced cellular invasion of
the basement membrane. Strongin et al.(27) described
a multistep process in which activated MT-MMP acts as a cell surface
TIMP-2 receptor. The activated MT-MMPTIMP-2 complex in turn acts
as a receptor for progelatinase A by binding the carboxy end domain of
secreted progelatinase A.
Based on thrombin's known proliferative effects on various cell types (28) , including endothelial cells(29) , and the subsequent activation of MMPs occurring during angiogenesis, we hypothesized that thrombin may, in part, be responsible for the activation of endothelial cell progelatinase. We now demonstrate that thrombin treatment of endothelial cells results in the activation of progelatinase A; this occurs independently of signal transduction events mediated by the G-protein-coupled thrombin receptor, but is dependent on the presence of an uncharacterized plasma membrane activation mechanism. We propose that thrombin generated during the hemostatic process induces remodeling of the endothelium as a component of neoangiogenesis.
Recombinant human
progelatinase A, NH-terminal gelatinase (truncated
COOH-terminal domain deletion mutant of progelatinase A lacking amino
acids 418-631 (36 kDa)(18) , COOH-terminal gelatinase A
lacking amino acids 1-414 (functionally inactive 31 kDa), an
inactive recombinant progelatinase A mutant with glutamic acid residue
(E
) replaced by alanine
(proE
->A) capable of binding TIMP-2(30) ,
recombinant TIMP-1 and TIMP-2(18) , and CT1399 (N
-hydroxy-N
-[1-(S)-(morpholiosulfonylaminoethyl-aminocarbonyl)-2-cyclohexylethyl]-2-(R)-(4-chlorophenylpropyl)succinamide)
were gifts from Dr. A Docherty (Celltech Ltd., Slough, United Kingdom).
For functional expression studies of the
thrombin receptor, the full-length construct encompassing the thrombin
receptor open reading frame (4) was cloned into the BamHI site of pcDNAI (Invitrogen Corp., San Diego, CA) which
contains the neomycin phosphotransferase cassette. Rat-2 fibroblasts
were plated at a density of 5 10
cells/100-mm
dish and transfections completed using 20 µg of individual
plasmid constructs and calcium phosphate precipitation(31) .
After 2-3 days, complete media was supplemented with 0.5 mg/ml
geneticin (G418, Life Technologies, Inc.) and changed every 3 days.
Individual colonies were subsequently ring-cloned, pooled, and
evaluated for functional thrombin receptor expression by
microspectrofluorimetry. Briefly, confluent cells were propagated on
glass coverslips and serum starved for
18 h. Cells were then
loaded with 2.5 µM fura 2/AM for 60 min at 37 °C,
rinsed with phosphate-buffered saline, and placed in a Leiden
temperature-controlled chamber. Individual cells were optically
isolated using an inverted microscope equipped with quartz optics and
alternatively illuminated at wavelengths of 340 and 380 nm (Photon
Technologies International, Trenton, NJ). Measurements of fluorescent
intensity were performed at a rate of 20 points/s.
Western blotting was performed using affinity-purified rabbit polyclonal antibodies directed against purified native proteins (TIMP-1, TIMP-2) and a synthesized peptide corresponding to the sequence of gelatinase A (amino acids 67-89) as described previously (35) . Recombinant proteins were used as positive controls in immunoblotting.
Quantitation of gelatinase A (both latent and activated enzyme detected), stromelysin-1, interstitial collagenase, TIMP-1, and TIMP-2 was performed on HUVEC-conditioned media using enzyme-linked immunosorbent assays as described previously(36, 37) .
Assay for the degradation of soluble gelatin was performed using rat
skin type I collagen (100 µg/assay) labeled in vitro with
[H]formaldehyde and heat-denatured as described
previously(33) . In vitro activation of progelatinases
was performed by 2 h of incubation of cell-conditioned media with 1
mM APMA(33) .
The proteolytic effects of 100 nM thrombin on recombinant TIMP-1 and TIMP-2 were tested by 18 h of incubation of proteins at 37 °C. Protein bands were examined by SDS-PAGE followed by Coomassie Blue staining.
Protein determinations were made using the bicinchoninic acid reagent as per manufacturer's instructions (Pierce BCA Protein Assay Reagent).
Statistical differences between groups were performed using the analysis of variance.
Figure 1:
A,
gelatin zymogram showing a dose-response curve of the effect of
thrombin on release and activation of endothelial gelatinases.
Approximately 7 10
cells (HUVEC) were propagated on
gelatin-coated wells, serum starved for 4 h, and then activated for
various time points with distinct concentrations of thrombin in a final
volume of 200 µl. A 20-µl aliquot was then evaluated by gelatin
zymography as outlined under ``Experimental Procedures.'' Lanes 1-3 contain the supernatants from cells grown in
M199 media alone; lanes4-6 had 10 nM thrombin added; lanes7-9 had 100
nM thrombin added. Lanes1, 4, and 7 contain cell supernatants collected over 4 h of incubation; lanes2, 5, and 8 were supernatants
collected over 8 h; lanes3, 6, and 9 contain supernatants collected over 24 h. As noted in the figure,
thrombin accelerated the activation of 72-kDa progelatinase A to 64 and
62 kDa gelatinolytic bands progressively over 24 h. Activation of
progelatinase A was noted within 2 h (data not shown). Molecular masses
of gelatinolytic bands are represented on the left and are
based on protein standards run simultaneously (myosin, 200 kDa;
-galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine
serum albumin, 66 kDa; ovalbumin, 42 kDa). B, densitometric
analysis of thrombin-induced activation of endothelial progelatinase A.
Laser densitometry (gelatin zymogram in Fig. 1A) was
employed to quantitate change in latent gelatinase A (72 kDa) and
activated gelatinase A (combined 64 and 62 kDa) following the addition
of thrombin or buffer to HUVEC over 24 h. At the lower end of the
linear range of gelatin digestion, the densitometry readings are
linear; however, the lysis bands are barely perceptible by eye and do
not photograph well(32) .
Figure 4:
Gelatin zymograms demonstrating the effect
of serine, cysteine, and aspartate proteinase inhibitors and of a
protein synthesis inhibitor on thrombin-mediated activation of
progelatinase A in endothelial cells. HUVEC were incubated at 37 °C
with or without thrombin; proteinase inhibitors were added to
endothelial cells 1 h prior to addition of thrombin. Conditioned media
was collected after 18 h. A demonstrates the effect of
cycloheximide, soybean trypsin inhibitor, aprotonin, E-64, pepstatin,
COOH-terminal gelatinase A, and NH-terminal progelatinase A
on thrombin-induced endothelial cell activation of progelatinase A.
HUVEC were incubated for 18 h without (CONT, lane1) or with 20 nM thrombin (lanes2-9) in the presence of 1 µM cycloheximide (CYCL, lane3), 2
µM soybean trypsin inhibitor (SBTI, lane4), 1 µM aprotonin (APR, lane5), 1 µM E-64 (lane 6), 1
µM pepstatin (PEPS, lane7),
0.3 µM COOH-terminal gelatinase A (C-GL, lane8), and 0.3 µM NH
-terminal
gelatinase A (N-GL, lane9). B demonstrates the activation of progelatinase (lane1, no thrombin added) by the addition of thrombin (20
nM) to endothelial cells (lane2). PPACK (2
µM) inhibited progelatinase A activation by 80%;
2-antiplasmin (
AP, 1 µM)
had no inhibitory effect on activation.
To document the class of proteinases identified on zymography, the gels were incubated with low molecular weight proteinase inhibitors (Fig. 2A). Incubation of the gelatin-impregnated gels with 1,10-phenanthroline or EDTA (chelators of metal ions) completely blocked the appearance of the endothelial-conditioned media gelatinolytic bands at 200, 92, 72, 64, and 62 kDa; PMSF (inhibitor of serine proteinases), leupeptin (inhibitor of serine proteinases and cysteine proteinases), and N-ethylmaleimide (inhibitor of cysteine proteinases) had no effect on these lytic bands. This inhibition profile demonstrates that these gelatinolytic bands are bona fide metalloproteinases.
Figure 2: A, effect of proteinase inhibitors on the development of gelatinolytic bands in zymograms. Lanes1, 3, 5, 7, 9, and 11 contain latent and activated gelatinase A and activated gelatinase B isolated from human plasma (included as a positive control for gelatinases)(35) ; lanes 2, 4, 6, 8, 10, and 12 contain gelatinases released by endothelial cells in the presence of 10 nM thrombin during 18 h of incubation. Following electrophoresis of specimens and removal of SDS by washing the gel in 2.5% Triton, the gel was incubated for 48 h as follows: lanes1 and 2 with buffer alone; lanes3 and 4 with 25 mM EDTA; lanes5 and 6 with 1 mM 1,10-phenanthroline; lanes7 and 8 with 1 mM leupeptin; lanes9 and 10 with 2 mM PMSF; and lanes11 and 12 with 3 mMN-ethylmaleimide. B, immunoblot analysis of conditioned media from thrombin-treated human umbilical endothelial cells. Specific rabbit antibodies to human gelatinase A (GLA, lanes1 and 2), TIMP-2 (lanes3 and 4), and TIMP-1 (lanes5 and 6) were employed. Lanes1, 3, and 5 contain 1-3 µg of recombinant gelatinase A, TIMP-2, and TIMP-1 respectively. Lanes2, 4, and 6 contain conditioned media from 10 nM thrombin-treated endothelial cells (27 µg protein/lane).
To confirm the identification of the gelatinolytic bands released by thrombin-treated endothelial cells, progelatinase A (72 kDa) and activated gelatinase A (64-62 kDa) were identified on immunoblots using specific rabbit anti-human antibodies (Fig. 2B). Additional gelatinase A immunoreactive bands were noted at 52, 44, and 30 kDa which is consistent with inactive species of gelatinase A (no gelatinolytic activity on zymography) as described previously(40) . TIMP-2 immunoreactive bands were noted at 21, 62, and 72 kDa which is consistent with free TIMP-2, TIMP-2 bound to activated gelatinase A, and TIMP-2 bound to latent gelatinase A, respectively(35) . TIMP-1 immunoreactive bands were noted at 28 and 62 kDa which is consistent with free TIMP-1 and TIMP-1 bound to activated gelatinase A, respectively.
A direct method for examining
the degree of gelatinase activation was to incubate conditioned media
(20 concentrated) from non-treated endothelial cells versus thrombin-treated cells with [
H]gelatin for 2
h. As noted in Fig. 3, conditioned media from untreated HUVEC
degraded 0.2 ± 0.1% gelatin/mg protein; preincubation of this
conditioned media with APMA (an activator of MMPs) resulted in 433
± 34% gelatin degradation/mg. In contrast, conditioned media of
thrombin-treated endothelial cells degraded 374 ± 28% gelatin/mg
protein (p < 0.001 as compared to non-thrombin treatment);
preactivation of this conditioned media with APMA resulted in 466
± 69% substrate degradation/mg indicating that most of the
gelatinase A was activated after incubation of HUVEC with thrombin.
Incubation of conditioned media from non-thrombin-treated endothelial
cells with 100 nM thrombin for an additional 24 h (in absence
of cells) resulted in minimal enhancement of substrate degradation (p > 0.2). This result indicates that endothelial cells are
essential in thrombin-induced activation of progelatinase A. The
ability of TIMP-1 to inhibit the functional/biological activity of
activated gelatinase A was also examined. Incubation of APMA-activated
conditioned media from thrombin-treated and -untreated cells with
TIMP-1 resulted in almost total inhibition of substrate degradation.
Figure 3:
Histogram showing the effect of thrombin
treatment of endothelial cells on activation of progelatinase as
measured in a soluble [H]gelatin degradation
assay. Conditioned media was collected from buffer-treated HUVEC (lefthandpanel) and 100 nM thrombin-treated
HUVEC (righthandpanel) after 18 h of incubation in
M199 media. Conditioned media was concentrated from untreated cells
(final protein concentration 1343 µg/ml) and thrombin-treated cells
(1357 µg/ml), incubated with [
H]gelatin (100
µg/ml) for 2 h and [
H]gelatin degradation was
measured as described under ``Experimental Procedures.''
Degree of substrate degradation is expressed as percent gelatin
degradation/milligram of protein, to account for differences in protein
concentration between samples. Thrombin-induced endothelial cell
progelatinase A activation was highly significant as compared to
buffer-treated cells (p < 0.001). Addition of thrombin to
conditioned media of untreated cells (no cells present; lefthandpanel) did not result in significant activation of
progelatinase A. APMA was used to activate latent
MMPs.
Figure 5:
Gelatin zymogram displaying the effect of
modulation of the thrombin receptor on progelatinase A activation in
endothelial cells. Experiments were completed as outlined in Fig. 1, and conditioned media was tested for gelatinolytic
bands. HUVEC were incubated for 18 h at 37 °C with media only (lane1), 20 µg/ml and 100 µg/ml
TR (thrombin receptor-activating peptide, lanes2 and 3, respectively). Lanes4-6 were derived from endothelial cells treated
with 10 nM thrombin. 400 µg/ml anti-TR
(AbTR, lane5) and hirudin (HR, 1 unit/ml: lane6) were added to HUVEC
for 1 h prior to the addition of thrombin. Molecular weights are
represented on the left.
Figure 6: Gelatin zymogram showing the effect of inhibitors of matrix metalloproteinases on thrombin-mediated activation of progelatinase A. A, demonstrates the effect of a hydroxamic acid-based inhibitor of MMPs, CT1399 (Inhibitor-CellTech Ltd.), on activation of HUVEC progelatinase A. HUVEC were incubated without thrombin (lane1) and with 20 nM thrombin (lanes2-4). CT1399 was added in doses of 10 and 100 nM (final concentration) to the HUVEC cell incubations, lanes3 and 4, respectively, 1 h prior to the addition of thrombin. After 18 h of incubation, the conditioned media was collected and tested. B, demonstrates the more potent inhibitory effect of TIMP-2, as compared to TIMP-1, on thrombin-induced activation of HUVEC progelatinase A. Lane1 represents gelatinases released by HUVEC during 18 h of incubation in the absence of thrombin. Lanes 2-6 represent 18-h conditioned media from endothelial cells incubated with 20 nM thrombin. Lanes3 and 4 represent 18 h conditioned media from thrombin-stimulated cells which were preincubated with recombinant TIMP-1 (24 and 960 nM, respectively). Lanes5 and 6 represent 18 h of conditioned media from thrombin-stimulated cells which were preincubated with recombinant TIMP-2 (24 and 960 nM, respectively).
Figure 7:
Functional expression of the thrombin
receptor (TR) on Rat-2 fibroblasts. Rat-2 cells transfected
with thrombin receptor cDNA (Rat-TR) and untransfected cells (Rat-2)
were propagated on coverslips, loaded with fura 2/AM for 60 min, and
evaluated by microspectrofluorimetry (A). Rat-TR cells
displayed typical elevations in cytosolic calcium when activated
(identified by arrow) by 10 nM thrombin (a)
or 20 µM TR (b). No
responses are seen with Rat-2 cells activated by 10 nM thrombin (c). B, the lack of effect of thrombin
on activation of progelatinase A in Rat-TR is demonstrated. Rat-2 cells
and Rat-TR cells were cultivated in serum-free media with or without
thrombin. Lanes1, 2, and 5 contain
conditioned media collected after 24 h of incubation in M199 of parent
Rat-2 cells not transfected with TR cDNA. Lanes3, 4, and 6 contain conditioned media of Rat-TR cells
expressing the thrombin receptor. Lanes1, 3, and 5 contain conditioned media of untreated
cells; lanes2, 4, and 6 contain
conditioned media supplemented with 40 nM thrombin. Lanes5 and 6 contain conditioned media, which
following separation from cells, was subsequently treated for 2 h with
1 µM APMA to active progelatinase A. The 68 and 70 kDa
doublet band of gelatinolytic activity is characteristic of rat
progelatinase A. Metalloproteinase activation with APMA generated the
characteristic 62-kDa active gelatinase A.
Based on the well known capacity of TIMP-2, but not TIMP-1, to bind to the COOH-terminal domain of progelatinase A (stabilization site) and inhibit membrane induced activation of progelatinase A(17) , we compared the effect of these inhibitors on thrombin activation of MMPs. Addition of TIMP-2 (24 nM final concentration) to endothelial cell cultures was able to totally abrogate the activation of 72-kDa progelatinase A to 64 and 62 kDa gelatinolytic bands. TIMP-1 (24 nM) had no inhibitory effect on progelatinase A activation; a 40-fold higher concentration of TIMP-1 (960 nM) was required to produce 55% inhibition of progelatinase A activation (Fig. 6B). TIMP-2 (24 nM), but not TIMP-1 (24 nM), also inhibited spontaneous activation of progelatinase A (data not shown).
Figure 8:
Northern analysis of MT-MMP. A,
10 µg of total cellular RNA was size-fractionated in a 1%
denaturing agarose gel, transferred to nylon membranes, and incubated
with 1.7 kilobases (kb) of
P-radiolabeled
MT-MMP cDNA as probe. Lanes1-5 were washed
under high stringency wash conditions, and lanes6 and 7 were washed under low stringency wash conditions
(see ``Experimental Procedures''). Both blots were analyzed
by autoradiography after a 3-day exposure. The integrity and relative
quantities of RNA were documented by stripping the blots and reprobing
with a human actin cDNA probe (data not shown). A single
4.5-kilobase mRNA transcript corresponding to the known MT-MMP
band (arrow) is readily detectable in HUVEC, HEL cells, and
Rat-2 fibroblasts. Size markers corresponding to the 28 S and 18 S
ribosomal bands are indicated to the left. B, Western
blot analysis for MT-MMP of cell extracts from human umbilical vein
endothelial cells and Rat-2 fibroblasts. Cell extracts were prepared by
1% SDS treatment of washed cells. SDS-PAGE of cell extracts was
followed by immunoblotting with mouse monoclonal antibodies
(113-5B7) to MT-MMP. Lanes1-3 contain
30-60 µg protein/lane of cell extract from wild-type Rat-2
cells, Rat-2 fibroblasts expressing functional thrombin receptor and
HUVEC, respectively. The marker on the right identifies MT-MMP
at 63 kDa. The faint bands at 70 and 103 kDa presumably represent
nonspecific staining. Molecular weight marker proteins are identified
on the left.
MT-MMP protein was identified by immunoblotting in both HUVEC and Rat-2 fibroblast lysates as a 63-kDa protein using monoclonal antibody 113-5B7 (Fig. 8B). Staining intensity of MT-MMP bands were equivalent between wild-type and TR-transfected Rat-2 cells. The 63-kDa MT-MMP protein was also identified in HUVEC and Rat-2 cells using a rabbit polyclonal antibody to MT-MMP (data not shown).
It has been proposed that proteinase activity is involved at three discrete points during angiogenesis: 1) local degradation of basement membrane allowing migration of endothelial cells out of the existing vessel, 2) migration of endothelial cells through stroma, and 3) remodeling of the basement membrane as the new vessel forms(12) . Matrix metalloproteinases and plasminogen activators appear to have important roles in these remodeling events. Early studies indicated that endothelial cells secrete relatively high concentrations of immunoreactive, but functionally inactive metalloproteinases (41) . Difficulty in demonstrating metalloproteinase activity was attributed to endothelial cell production of large amounts of TIMP-1 and TIMP-2 which form complexes with and inhibit MMP activity. Endothelial cells secrete gelatinase A and gelatinase B selectively in a basal direction further supporting a role for these proteinases in turnover of basement membrane components during angiogenesis(9) . Experiments examining the function of gelatinase A in endothelial cell tube formation demonstrated that exogenous TIMP-1 or TIMP-2 were able to inhibit and exogenous gelatinase A was able to stimulate vessel formation, thereby emphasizing the requirement for balanced production of gelatinases and inhibitors in the early stage of angiogenesis(12, 14, 42) . Specific pharmacologic inhibitors of MMPs have also been demonstrated to decrease angiogenesis in experimental animals(43) .
Based on
the central role of thrombin in diverse aspects of endothelial cell
function(2, 4, 5) , and the potential
importance of gelatinases in
angiogenesis(12, 14, 39, 41, 42) ,
we considered the possibility that thrombin may be involved in the
regulation of matrix metalloproteinases in endothelial cells. We herein
report that within 2-4 h of addition of thrombin to endothelial
cells, increased amounts of activated gelatinase A were detected in
conditioned media; progelatinase B was not activated under these
conditions. The total concentration of gelatinase A (latent plus
activated), released by endothelial cells as measured by a specific
immunoassay, was not significantly increased by thrombin treatment
suggesting that thrombin initiates the progelatinase A activation
mechanism without affecting progelatinase A synthesis. This was
confirmed by showing that treatment of endothelial cells with
cycloheximide, an inhibitor of protein synthesis, did not prevent
activation of progelatinase A, although it did decrease total
gelatinase A production. In addition, a 200-kDa gelatinase was
induced by thrombin treatment of HUVEC. A similar 200 kDa gelatinolytic
band has been identified in human plasma (35) (see Fig. 2A) and in the conditioned media from cytokine
treated endothelial cells (39) and keratinocytes(44) .
To explore the mechanism of thrombin activation of progelatinase A,
we pretreated HUVEC with proteinase inhibitors prior to the addition of
thrombin to endothelial cell cultures. Pretreatment with PPACK or
hirudin, specific inhibitors of thrombin, abrogated thrombin-induced
activation of progelatinase A indicating that the proteolytic activity
of thrombin is an essential component of the process. To determine if
the thrombin effect is mediated through the recently described
functional thrombin receptor(4) , the anti-thrombin receptor
antibody (4) , which inhibits numerous endothelial cell
responses to thrombin, was tested; neither this antibody nor the
thrombin receptor activating peptides TR or
TR
displayed effects on endothelial cell
activation of progelatinase A. These results indicate that the
thrombin-tethered ligand receptor mechanism is not involved in
activation of progelatinase A, which lends support to the possible
existence of a second unidentified cell receptor for thrombin (4) . To more specifically evaluate the role of the thrombin
receptor in progelatinase activation, the cDNA encompassing the open
reading frame of TR was stably transfected into Rat-2 fibroblasts.
Addition of thrombin to transfected cells resulted in the anticipated
calcium flux response, but not activation of progelatinase A,
suggesting that activation of progelatinase A occurs independent of
this signal transduction event mediated by the thrombin receptor.
The observation that thrombin is unable to activate recombinant
progelatinase A in a cell-free system (16) indicates that the
endothelial cell is an essential component of the thrombin-induced
progelatinase A activation mechanism. Two types of data from our
experiments support the hypothesis that the endothelial plasma membrane
is responsible for activation of progelatinase A: 1) addition of 24
nM TIMP-2 (20-fold excess compared to endogenous secreted
TIMP-2), but not 24 nM TIMP-1, to endothelial cells abrogated
thrombin-induced activation of progelatinase A; and 2) addition of the
COOH-terminal domain of gelatinase A, but not a progelatinase A mutant
lacking the COOH-terminal domain(30) , abrogated thrombin
activation of endothelial progelatinase A. The inhibitory effect of 24
nM TIMP-2, but not TIMP-1, is explained by the fact that
excess TIMP-2 binds to latent gelatinase A near the COOH-terminal
domain of the molecule producing a stabilized complex which may
interfere with the interaction between the plasma membrane and latent
gelatinase A required for the activation of progelatinase
A(18, 45) . Similarly, the competitive inhibitory
effect on progelatinase A activation of the intact COOH-terminal region
of gelatinase A, but not a mutant gelatinase A with a deleted
COOH-terminal domain, suggests that binding of the COOH-terminal region
of gelatinase A to the plasma membrane activator mechanism is required
for metalloproteinase activation. Strongin et al.(20) have also demonstrated that the plasma
membrane-dependent activation of 72-kDa progelatinase A is followed by
conversion to a 64 kDa intermediate and subsequently a 62-kDa active
enzyme. Formation of a complex between progelatinase A and TIMP-2
inhibited activation to the 64 kDa intermediate. The lack of inhibitory
effect of low dose TIMP-1 may be due to the fact that TIMP-1 can only
bind to activated gelatinase A at a site closer to the
NH
-terminal domain of gelatinase A and cannot bind to the
COOH-terminal region of latent gelatinase A. Of relevance to this
discussion, Murphy et al.(13) demonstrated that
TIMP-2, but not TIMP-1, inhibited proliferation of endothelial cells
and may limit neovascularization through this mechanism.
Recent data
of Strongin et al.(27) suggest that activation of
progelatinase A requires the assembly of a trimolecular complex on the
cell surface. According to this hypothesis, endothelial cell secretion
of TIMP-2 in low concentration which does not excede the receptor
capacity of MT-MMP, is followed by cell surface binding of TIMP-2 to
MT-MMP, and subsequent binding of the COOH-terminal domain of the
progelatinase A molecule to the TIMP-2MT-MMP complex. Generation
of this trimolecular complex culminates in activation of progelatinase
A. Our Northern and Western blotting data, demonstrating expression and
synthesis of MT-MMP by endothelial cells, is consistent with a role for
MT-MMP in progelatinase A activation. Inhibition of protein synthesis
with cycloheximide, however, was not accompanied by abrogation of
thrombin-induced activation of progelatinase A, suggesting that in
endothelial cells the activation mechanism does not require induction
of MT-MMP synthesis. Our demonstration of MT-MMP expression and
synthesis by Rat-2 fibroblasts which lack a thrombin-induced plasma
membrane activation mechanism for progelatinase A further suggests that
other factors are required for cell surface activation of progelatinase
A. Whether serine proteinases such as plasmin and possibly thrombin are
involved in the proteolytic processing of endothelial 63-kDa MT-MMP to
the 60 kDa activated form as suggested by Strongin et al.(27) , will require additional study.
In conclusion, these experiments indicate that endothelial cells and smooth muscle cells (data not shown) possess a unique capacity for thrombin-induced activation of progelatinase A that may facilitate angiogenesis.