From the Division of Vascular Surgery, Department of Surgery, Dartmouth Medical School, Dartmouth College, Hanover, New Hampshire 03756
Received for publication, July 19, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Plasminogen activator inhibitor-1 (PAI-1) is a
serpin protease inhibitor that binds plasminogen activators (uPA and
tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the
active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted
into one of the beta sheets, thus making the reactive center loop
unavailable for interaction with the plasminogen activators. We
truncated porcine PAI-1 at the amino and carboxyl termini to eliminate
the reactive center loop, part of a heparin binding site, and a
vitronectin binding site. The region we maintained corresponds to amino
acids 80-265 of mature human PAI-1 containing binding sites for
vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the
helix F region. The interaction of "inactive" PAI-1,
rPAI-123, with plasminogen and uPA induces the
formation of a proteolytic protein with angiostatin properties.
Increasing amounts of rPAI-123 inhibit the proteolytic
angiostatin fragment. Endothelial cells exposed to exogenous
rPAI-123 exhibit reduced proliferation, reduced tube
formation, and 47% apoptotic cells within 48 h. Transfected
endothelial cells secreting rPAI-123 have a 30% reduction
in proliferation, vastly reduced tube formation, and a 50% reduction
in cell migration in the presence of VEGF. These two studies show that
rPAI-123 interactions with uPA and plasminogen can inhibit
plasmin by two mechanisms. In one mechanism, rPAI-123
cleaves plasmin to form a proteolytic angiostatin-like protein. In a
second mechanism, rPAI-123 can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.
Plasmin, the end product of plasminogen activator-derived
proteolysis, is a trypsin-like enzyme that functions in the degradation of fibrin and extracellular matrix
(ECM)1 proteins. Plasminogen
is converted to plasmin by the activity of urokinase plasminogen
activator (uPA) or tissue plasminogen activator (tPA) (1, 2).
Plasminogen, uPA, and plasmin bind to uPA receptors (uPAR) localizing
the proteolytic activity of plasmin to the cell surface where it is
used by migrating cells to degrade the ECM and basement membrane
proteins (1, 3, 4).
The proteolytic activity of plasmin is regulated by plasminogen
activator inhibitors 1 and 2 (PAI-1 and PAI-2). PAI-1 and uPA are
coordinately expressed in the normal regulatory pathways of plasminogen
activator-derived proteolysis.
PAI-1 is a secreted protein that is rapidly converted to a latent form
at physiological temperatures. It becomes stabilized and activated when
it binds vitronectin (Vn) (5-7), a cell adhesion glycoprotein (6, 8)
found primarily in the ECM at acute injury, in tissue repair (9), and
in some malignant tumors (10).
PAI-1 is a multifunctional regulatory protein. Vitronectin-bound PAI-1
(active) plays a crucial role in regulating the proteolytic degradation
of the extracellular matrix, which in turn regulates cell migration and
invasion (5, 11). Active PAI-1 regulates proteolysis by inhibiting
uPA/tPA. Upon binding Vn, activated PAI-1 then binds a
uPA·uPAR complex, thus forming a PAI-1·uPA·uPAR complex (12-14).
These interactions result in a cascade of regulatory events: 1)
dissociation of PAI-1 and uPAR from their respective vitronectin
binding sites (15, 16); 2) inactivation of uPA and PAI-1 by
internalization and lysosomal degradation of the PAI-1·uPA complex
(17, 18); 3) recycling of uPAR to a new site on the cell surface (19);
4) availability of PAI-1 binding sites on vitronectin for attachment of
other molecules, such as PAI-1 belongs to the serpin family of serine protease inhibitors. The
PAI-1 protein contains a strained loop reactive center, amino acids
320-351, located at the carboxyl terminus of the molecule (25-29).
PAI-1 initially interacts with uPA at Arg-346 (30) and acts as a
pseudo substrate "bait." However, uPA is unable to cleave the
pseudo substrate and PAI-1/uPA form a stable complex rendering uPA
inactive (29, 31-34). It has been demonstrated that the PAI-1-reactive loop must then insert between strands 3 and 5 of the The region of PAI-1 distant from the reactive center interacts with
other molecules involved with the proteolytic and fibrinolytic pathways. The PAI-1 interactive sites for vitronectin are at Gln-55, Phe-109, Met-110, Leu-116, Gln-123, and residues 128-145 (43), heparin
(Lys-65 to Lys-88) (44), a second tPA site (amino acids 128-145) (43),
and thrombin interaction at residues 128-145 (7, 30). The region
between amino acid residues 110-145 binds urokinase and fibrin (30).
The helix F of PAI-1 (residues 127-158) is thought to play an
important role in function and stability by controlling the rate of
transition from active to latent conformation (45).
In an attempt to gain a better understanding of the relationship
between PAI-1 structure and its multiple regulatory functions, the
porcine PAI-1 gene was systematically dissected to obtain truncated PAI-1 proteins, rPAI-1. The rPAI-1 proteins would be used as
tools to identify the regions of the inhibitor that bind (complex)
other important regulatory molecules in proteolytic and adhesion
mechanisms. In so doing, we hoped to identify other domains that have a
biological function.
We have closely examined one deleted PAI-1 gene product
(rPAI-123) that encompasses the porcine PAI-1
gene sequences (46) containing nucleotides 444-999 corresponding to
nucleotides 238-793 in the human PAI-1 gene (amino acids
80-265 of mature protein) (47). The recombinant rPAI-123
fragment: 1) excluded those sequences coding for the reactive center
loop at the carboxyl terminus; 2) eliminated a vitronectin binding
site, more than one half of a heparin binding region, and the latency
residue at Lys-323 (42); 3) retained vitronectin and a second tPA
binding region at amino acids 128-145, part of a heparin binding site
at amino acids 80-88, a thrombin interactive region at residues
115-148, residues 127-158, which contain the helix F and the loop
that connects helix F with helix s3A (48, 49).
The data we present show that when a truncated PAI-1 protein,
rPAI-123, complexes with plasminogen or plasminogen and
uPA, the following results occur: 1) Plasmin is cleaved to produce a
proteolytic angiostatin-like cleavage product. UPA enhances the
formation of the angiostatin-like protein by increasing the amount of
available plasmin. 2) Excess rPAI-123 inhibits proteolytic angiostatin formation by binding to uPA and plasminogen such that they
are not available to form plasmin that can be cleaved into angiostatin.
3) Cultured endothelial cells exposed to rPAI-123 exhibit a
decrease in proliferation, a loss in tube formation capability,
increased apoptosis, and decreased migration in the presence of
VEGF.
Isolation of Porcine rPAI-123 Gene Fragment--
The
555-bp DNA fragment coding for the truncated rPAI-123
protein (Fig. 1) was isolated from
porcine aortic endothelial cells by reverse-transcribing RNA into
cDNA using the manufacturer's random primer and protocol (Roche
Molecular Biochemicals, Indianapolis, IN). The cDNA was made
double-stranded in a PCR reaction containing the following porcine
PAI-1 primers: 5'-primer, 5'-GGAATTCAAGGAGCTATGG-3'; 3'-primer,
5'-GCTCTAGATTTCCACTGGCTGATG-3'. The PCR conditions were as follows:
~1 pg of the PAI-1 gene was combined with 50 pmol of the
5'- and 3'-primers and amplified at 95 °C, 10 min; 58 °C, 1 min;
Taq DNA polymerase (5 units) was added; then 72 °C, 1 min
followed by 34 cycles of 95 °C, 1 min; 58 °C, 1 min; 72 °C, 1 min.
The PAI-1 DNA fragments were double-digested with EcoRI and
XbaI and ligated into a Pischia pastoris yeast
shuttle vector, pGAPZ rPAI-123 Protein Expression and Isolation--
The
DNA from a positively identified ligation product containing the
rPAI-123 gene fragment was linearized at a specific restriction enzyme site in the pGAPZ PAI-1 Activity Assay to Detect Plasmin Formation--
The
rPAI-123 protein (30 nM) was incubated with 10 nM of uPA (American Diagnostica Inc., Greenwich, CT) in
TBS, pH 7.8, containing 100 µg/ml BSA and 0.01% Tween 20 at room
temperature for 1 h. Following the PAI-1·uPA reaction, 33 nM plasminogen and 15 µg/ml Chromozym PL (Roche
Molecular Biochemicals) were added to the rPAI-123·uPA
reaction mix. The reactants continued to incubate at 37 °C until
color change could be detected in the uPA standards. The colorimetric
change was measured at 405 nm on an EL-311 Microplate Autoreader
(Bio-Tek Instruments, Burlington, VT). In this assay plasmin cleaves
Chromozym PL resulting in a p-nitraniline molecule that
absorbs at 405 nm. The colorimetric change is a measurement of plasmin formation.
Zymography Functional Assay--
1.3% casein was incorporated
into an SDS-polyacrylamide separating gel. The casein serves as a
substrate for plasmin proteolytic activity. The rPAI-123
protein (30-120 nM) was bound to 10 nM of uPA
at room temperature for 1 h. Following this reaction, 33 nM of plasminogen was added to the
rPAI-123·uPA reaction mix and incubated at 37 °C for
20-30 min. The samples were electrophoresed at constant voltage on a
polyacrylamide gel containing 1.3% casein. The polyacrylamide gels
containing the electrophoresed samples were first washed in 2.5%
Triton-X before an overnight incubation at 37 °C in a 50 mM Tris-HCl buffer, pH 7.9. The gels were fixed in 40%
methanol, 10% acetic acid and then stained in 10% Coomassie Blue for
1 h. The gel was destained in 10% methanol, 10% acetic acid to
detect the regions of proteolysis (50, 51).
Zymography Analysis of Vitronectin Binding to
rPAI-123--
Vitronection (Sigma Chemical Co., St. Louis,
MO) at a concentration of 25 nM was incubated with 30 nM of rPAI-123 for 1-2 h at 37 °C before
performing the zymography functional assay as described above.
Western Blot Analysis of the 36-kDa Proteolytic Fragment
Resulting from rPAI-123, Plasminogen, uPA
Reaction--
Equal fractions of reaction mixtures for the zymography
assay described above were placed on an SDS, nonreducing, 4-20%
gradient polyacrylamide gel. The electrophoresed samples were
transferred to a nitrocellulose membrane. The membrane containing the
transferred proteins was blocked and washed. The results were
visualized on a Western blot probed with 1 µg/ml goat anti-human
plasminogen kringles 1-3 (angiostatin) (R&D, Minneapolis, MN) for
1 h at room temperature. The membrane was washed and blocked a
second time for 1 h at room temperature. A rabbit anti-goat IgG
(H+L) secondary antibody (Pierce, Rockford, IL) at a concentration of 1 µg/ml was incubated with the membrane for 1 h at room
temperature. A horseradish peroxidase-conjugated tertiary antibody
(donkey anti-rabbit IgG, Amersham Pharmacia Biotech, Arlington Heights,
IL) diluted 1:1000 amplified the binding reaction, which was ultimately
detected by addition of a chemiluminescent substrate (Amersham
Pharmacia Biotech) for 45 min at room temperature (52).
Varied Permutations for rPAI-123, uPA, and
Plasminogen Interactions--
The order in which rPAI-123,
uPA, and Plg were added to a reaction mixture was varied:
(a) uPA + Plg for 1 h at 37 °C, then rPAI-123 was added for an additional 1 h at 37 °C;
(b) rPAI-123 + uPA for 1 h at 37 °C,
then Plg was added for an additional 1 h at 37 °C; or
(c) rPAI-123 + Plg for 1 h at 37 °C,
then uPA was added for an additional 1 h at 37 °C.
In some cases, the amount of uPA and/or rPAI-123 was
varied. To ensure that either uPA or Plg was exhausted in a reaction mix, an additional amount of either uPA or Plg was added to some permutations The samples were electrophoresed on a zymogram as described.
Endothelial Cell Incubation with Exogenous
rPAI-123--
Porcine endothelial cells were plated at a
density of 0.5 × 106 cells/ml in 24-well culture
plates containing DMEM supplemented with 10% fetal calf serum. The
cells were allowed to adhere to the culture plate before adding
exogenous rPAI-123 (1 µg/ml). The cells were incubated at
37 °C for 24 h at which time an additional 1 µg/ml
rPAI-123 was added. This procedure was maintained for 5 days. Controls were endothelial cells alone or endothelial cells to
which equivalent amounts of secreted yeast cell protein were added. The
cells were observed for proliferation, attachment, and tube formation.
Insertion of rPAI-123 Gene into pCMV/Myc/ER
Eukaryotic Protein Expression Vector--
The 555-bp DNA fragment
coding for the truncated rPAI-123 was isolated from porcine
aortic endothelial cell RNA by reverse transcription as described
above. The cDNA was made double-stranded in a PCR reaction
containing the 3'-porcine PAI-1 primer described and a 5'-PAI-1 primer
to incorporate a PstI restriction enzyme site. The sequence
of the 5'-primer is 5'-AACTGCAGAAGGAGCTATGG-3'. The PCR conditions were
as described. The enzyme-activated PAI-1 DNA fragments were ligated
into a pCMV/Myc/ER eukaryotic protein expression vector (Invitrogen)
for amplification in E. coli. Isolates from each ligation
reaction were double-digested with restriction enzymes XbaI
and EcoRI to verify the presence of the gene insert (data
not shown). Positive isolates from the restriction enzyme digests were
verified by sequencing.
Electroporation of rPAI-123/pCMV/Myc/ER into
Endothelial Cells--
The pCMV/Myc/ER eukaryotic protein vector is
designed to provide directionality of protein expression to the
endoplasmic reticulum. The expressed protein can either be retained or
secreted by manipulation of a stop codon in the inserted gene of
interest. The rPAI-123/pCMV/Myc/ER DNA at a concentration
of 25-50 µg/400 µl DMEM supplemented with 10% FCS was placed in a
4-mm gap cuvette and electroporated into bovine endothelial cells using
a BTX Electro Cell Manipulator 600. The electroporation conditions were
550 V/capacitance and resistance, 1000 µF, 13 ohm, and a charging
voltage of 260 V. The pulsed cells were immediately transferred to a
culture plate containing DMEM supplemented with 10% FCS. Geneticin
(G418) at a concentration of 1 mg/ml was added to the cells 24 h
after electroporation.
Proliferation Assay--
Bovine endothelial cells stably
transfected with rPAI-123 were plated into a 24-well
culture plate at a density of 2.3 × 104/ml in DMEM
supplemented with 10% FCS. The cells were trypsinized and counted on a
hemocytometer plate at 24-h time intervals. Further verification of
proliferation was performed by use of the CellTiter 96 Aqueous One
Solution cell proliferation assay (Promega, Madison, WI) following the
manufacturer's protocol. In brief, 1 × 104
rPAI-123 transfected bovine endothelial cells were plated
into a 96-well microtiter plate in DMEM containing 10% FCS. After 24 or 48 h of growth, 20 µl of the manufacturer's supplied
proliferation reagent was added to each well and incubated at 37 °C
for 1-4 h. Absorbance was read at 490 nm in an EL-311 Microplate
Autoreader (Bio-Tek Instruments, Burlington, VT).
Tube Formation--
Bovine aortic endothelial cells stably
transfected with rPAI-123 were plated in 24-well plates at
a concentration of 1 × 105 cells/ml in DMEM
supplemented with 10% FCS. The cells were fed every 48 h and
maintained for a total of 21 days. Tube formation was observed. At day
14, a fluorescent ester probe, BCECF AM (Molecular Probes, Inc.,
Eugene, OR) was diluted to 10 mM in Me2SO. The
diluted fluorescent probe was added to the culture medium to obtain a final concentration of 5 µM and incubated for 60 min at
37 °C. Following the incubation, the cells were washed twice in
phosphate-buffered saline. The fluorescing cells were photographed with
a 35-mm camera at 200× magnification under a Nikon inverted microscope.
Apoptosis Detection--
Bovine aortic endothelial cells and
smooth muscle cells were each plated at a density of 1.0 × 106 cells/T-75 culture flask containing DMEM supplemented
with 10% fetal bovine serum. The cells were incubated for 24 h at
37 °C, 5% CO2 before adding either exogenous
rPAI-123 (1 µg/ml) or yeast supernatant protein (1 µg/ml). The cells were incubated an additional 18 h at 37 °C.
At which time the medium containing the detached cells was collected
and placed on ice while performing two HBSS washes. The medium and HBSS
washes containing the detached cells were combined, counted, pelleted,
and washed twice in cold phosphate-buffered saline. Fluorescein
isothiocyanate-conjugated ApopNexin and propidium iodide were added to
the cells following the manufacturer's protocol (Integren, Purchase,
NY) (53). Adherent cells were trypsinized, resuspended in DMEM
containing 10% fetal bovine serum, and incubated at 37 °C for 10 min. The pelleted cells were washed twice with cold HBSS. Fluorescein
isothiocyanate-conjugated ApopNexin and propidium iodide were
added to the pelleted cells. Each cell fraction was analyzed separately
on a FACScan (Becton Dickinson, San Jose, CA).
Migration Assay--
Endothelial cells stably transfected with
pCMV/Myc/ER/rPAI-123 or pCMV/Myc/ER were serum-starved
overnight in DMEM. The next day each transfected cell sample was
counted and 1 × 105 cells were plated onto a 24-well,
8-µm pore membrane (Corning, Inc., Corning, NY). Cells were allowed
to attach for 30 min. At that time, the top chamber containing the
membrane-seeded cells was placed into the bottom chamber of the
co-culture well that contained either DMEM supplemented with 10% FCS
or DMEM supplemented with 10% fetal bovine serum and VEGF (10 ng/ml).
The cells were allowed to migrate through the membrane pores for
4.5 h. The cells were fixed in 4% buffered formalin, stained with
toluidine blue, and mounted on glass slides. Five fields from each of
three test samples were counted at a magnification of × 200.
Isolation of Porcine PAI-1 Gene Fragments--
The porcine
rPAI-1 gene fragments rPAI-114 and
rPAI-123 were ligated and replicated in the P. pastoris protein expression vector. The double-digested DNA
isolated from selected colonies corresponded to their respective
molecular weight sizes of 1137 and 555 bp as shown in Fig. 2. The
sequences of human PAI-1 (huPAI-1) and porcine PAI-1 (poPAI-1) are 91%
homologous and have no "obvious functional differences" (46). The
rPAI-123 region of poPAI-1 contains 16 amino acid
variations from the huPAI-1 gene. The sequence we obtained
for rPAI-123 (data not shown) matches the corresponding sequences in the reported full-length poPAI-1 gene.
Protein Expression and Isolation--
Fig. 3 shows the
rPAI-123 protein that was isolated from the supernatant of
P. pastoris-transfected cells. The protein corresponds to
the appropriate molecular mass of 18.5 kDa.
Chromozym Assay to Detect Plasmin Formation--
The graph in Fig.
4 shows the results of the Chromozym
assay and compares huPAI-1 inhibition of uPA with uPA inhibition by rPAI-1 proteins rPAI-123 and rPAI-114
(full-length PAI-1). The rPAI-114 protein (lane
6) has activity equivalent to 36.6 IU/µg when compared with
huPAI-1 activity (rows 2 and 3). Interestingly, rPAI-123 (row 5) was able to inhibit ~50% of
plasmin formation when compared with the activity of the control
containing 0 IU of huPAI-1 (row 4). This equates to 8.25 IU
when compared with the measured 136 IU/0.44 µg of huPAI-1 (row
2). The rPAI23 protein lacks the reactive center loop
shown to be necessary for binding uPA to inhibit the activation of
plasmin. The numeric value ascribed to rPAI23 in this assay
is a measurement of plasmin formation, because color formation occurs
when the Chromozym PL is cleaved by plasmin.
Zymography Functional Assay--
The results of the zymography
experiments, which enable visualization of the functionality of
rPAI-123 (Fig. 5), clearly
show that the truncated PAI-1 protein, rPAI-123, inhibits
the 80-kDa proteolytic plasmin (the size of the uPA-cleaved
plasminogen) when incubated with either plasminogen (Fig. 5,
lanes 6-8) or uPA and plasminogen (Fig. 5, lanes
10-12). The degree of inhibition is dependent upon the uPA
concentration, but is nevertheless dramatically reduced when compared
with the plasmin formed with the equivalent concentration of uPA. In
all cases, a proteolytic cleavage product slightly greater than the
34-kDa molecular mass marker is visible. When the electrophoretic
separation is adjusted, the proteolytic product consists of two
distinct bands very close to each other that are ~36-38 kDa. The
degree of proteolysis associated with these fragments is substantial.
The proteolytic activity associated with the protein(s) is intensified
when rPAI-123 is first incubated with uPA and then
plasminogen (Fig. 5, lanes 10 and 11) as compared with the rPAI-123 and plasminogen reaction mixture (Fig. 5,
lanes 6 and 7). Increasing amounts (4-fold) of
rPAI-123 incubated with uPA ultimately inhibited the
proteolytic activity (lanes 8 and 12).
Furthermore, the inhibitory effect of full-length PAI-1
(rPAI-114) shown in lane 4 does not display
proteolytic cleavage products at 34 kDa as compared with the same
rPAI-123 reaction in lane 11.
Zymography Analysis of Vitronectin Binding to
rPAI-123--
PAI-1 has several vitronectin binding sites
and one known binding domain. A critical site within the amino terminus
of huPAI-1 is deleted in rPAI-123. However, more interior
Vn binding sites corresponding to huPAI-1 amino acids 109, 110, 116, and 128-145 have been maintained. In a folded mature PAI-1 protein,
the vitronectin binding regions are thought to be near each other. The
loss of one vitronectin binding site could effect the stability of
rPAI-123. Therefore, this experiment was performed to
analyze the formation of the 36- to 38-kDa proteolytic proteins when
rPAI-123 is associated with Vn. There is no significant
difference observed in the formation of the 36- to 38-kDa proteolytic
fragment when rPAI-123 is incubated with vitronectin (Fig.
6, lanes 9-12). We also
addressed the stability of rPAI-123 in various buffers
(TBS, TBS/Tween 20, TBS/BSA, TBS/Tween 20/BSA) and found no difference
that could be attributable to the buffer components with or without
vitronectin. If vitronectin is binding rPAI-123, then that
binding does not alter the functionality of rPAI-123.
Western Blot Analysis of the 36-kDa Proteolytic Fragment from
rPAI-123, Plasminogen, uPA Reaction--
Reaction samples
that were visualized on the functional zymogram (Fig. 5) were
electrophoresed on a 4-20% SDS, nonreducing polyacrylamide gel. The
proteins contained within the polyacrylamide gel were transferred to a
nitrocellulose membrane, and the Western blot was probed for
plasminogen kringles 1-3 (angiostatin). The results of the
angiostatin-probed membrane (Fig. 7)
correspond to the results seen on the zymogram (Fig. 5). In lanes
9 and 11 on both gels, the proteolytic proteins that
were inhibited by 120 nM of rPAI-123 are not
visible on the angiostatin probed membrane. However, lanes
8 and 10 clearly show the presence of those bands at lower concentrations (30 nM) of rPAI-123. It
should be noted that the angiostatin-probed protein band slightly above
the one that was eliminated by rPAI-123 remained intact.
Furthermore, note the lack of a positive angiostatin band in lane
4 that contains rPAI-114 protein (full-length
PAI-1).
Varied Permutations for rPAI-123, uPA, and Plasminogen
Interactions--
The varied permutations for rPAI-123,
uPA, and Plg interactions (Figs. 8-10) were designed to determine how
rPAI-123 induces the formation of proteolytic angiostatin.
Fig. 8 shows the results of the
experiment designed to test the effect of rPAI-123 on
plasmin formation. In Fig. 8, lanes 3 and
4, uPA and Plg were first reacted to form plasmin before the
addition of rPAI-123 (30 nM). The concentration of uPA in a reaction mixture was either 5 or 20 nM. In
lanes 5 and 6, uPA and 30 nM rPAI-123 were first reacted before the
addition of 33 nM of Plg. In all four lanes (lanes
3-6), the proteolytic angiostatin is formed at 36-38 kDa. The
plasmin at 80 kDa is nearly gone when compared with the plasmin in
lanes 1 and 2. Regardless of the permutation and
the concentration of uPA, any differences in the molecular mass or
intensity of the visible proteins represented in lanes
3-6 of the zymogram are negligible.
The effects of rPAI-123 on uPA formation of plasmin were
tested by adding rPAI-123 (120 nM) to uPA (5 nM) either before or after an incubation with Plg (30 nM). To ensure that all of the uPA or Plg was exhausted, a
second amount of uPA (5 nM) or Plg (30 nM) was
added for a third hour at 37 °C. The results of those experiments
are shown in Fig. 9. Lanes
5, 6, 9, and 10 represent uPA + 120 nM rPAI-123 combined in a reaction
for 1 h at 37 °C prior to adding Plg. In all cases, plasmin
formation is greatly reduced when compared with plasmin formed by uPA + Plg (lane 2), 30 nM rPAI-123 + uPA,
then Plg (lane 3), or uPA + Plg, then 30 nM
rPAI-123 (lane 4). Addition of more uPA
(lane 6) or more Plg (lane 10) does not increase
plasmin formation. In the samples where uPA + Plg occurred first before
the addition of 120 nM rPAI-123 (lanes 7, 8, 11, and
12), the amount of plasmin formed remains less than in the
uPA + Plg control samples (lane 2). In the
angiostatin-forming reaction in which 30 nM of
rPAI-123 is reacted with uPA and Plg (lanes 3 and 4), the amount of plasmin is much greater than either permutation with 120 nM of rPAI-123
(lanes 5, 6, 9, and
10).
In experiments that produced the data shown in Fig.
10, the concentration of
rPAI-123 (30 or 60 nM) and uPA (0.05-5.0
nM) were varied in the reaction mixes. In all samples where
rPAI-123 was added, it was first reacted with Plg before
the addition of uPA. In those samples (Fig. 10, lanes 6-9
and 11-14) the amount of plasmin that is formed is
substantially less than the plasmin produced in the controls,
lanes 2, 3, and 10. In
lanes 6 and 7, where the uPA is held
constant at 5 nM and the rPAI-123 concentration
is 30 nM, the amount of plasmin is slightly greater than in
lanes 8 and 9, where the amount of
rPAI-123 is 60 nM. In lanes
7 and 9, where excess Plg is added to the
reaction, there appears to be a slight increase in the level of plasmin
that is not seen in lane 9 that contains twice the amount of
rPAI-123. In lanes 11-14, where 30 nM of rPAI-123 was first reacted with Plg, then 0.5 nM uPA, the plasmin levels are reduced from that found
in lanes 6 and 10. When twice the
amount of rPAI-123 is added to Plg then 0.5 nM
uPA, the plasmin level is near background Plg control levels in
lane 1. Attention should be made to the low level of the
angiostatin fragment(s) seen in lanes 6-10.
Endothelial Cell Incubation with Exogenous
rPAI-123--
These experiments were performed to do an
initial observation of the effects of rPAI-123 on
endothelial cell growth, migration, and tube formation. The cell
cultures were allowed to grow for 5 days at which time tube formation
was already observable in both sets of control cells (Fig.
11A). The untreated control
endothelial cells and endothelial cells incubated with protein secreted
from yeast cells grew normally and became confluent (Fig.
11B). The endothelial cells grown in the presence of
exogenous rPAI-123 displayed characteristics very
dissimilar to the two controls as follows: 1) The cells easily became
detached in the first 1-2 days. Analysis of the
rPAI-123-treated endothelial and smooth muscle cells showed
47% of the combined detached and adherent endothelial cells were
apoptotic compared with 12% of apoptotic smooth muscle cells treated
with the same concentration of rPAI-123 (Fig.
11C). 2) The rate of proliferation was reduced 30% at the end of 96 h (Fig. 11D), and the cells never reached
confluency. 3) Tube formation did not occur in those endothelial cells
treated with exogenous rPAI-123 (Fig. 11B).
Effects of rPAI-123 Expression from Endothelial
Cells--
Expression and secretion of rPAI-123 from
endothelial cells was verified in a Chromozym Pl assay compared with
endothelial cells transfected with the empty vector and endothelial
cells overexpressing PAI-1 (54). The medium from each cell culture was
collected and assayed. The medium containing rPAI-123
contained 0.04 IU/ml plasmin as compared with undetectable plasmin in
the endothelial cells overexpressing PAI-1 and 0.5 IU/ml in medium from
endothelial cells transfected with the empty vector (data not shown).
Tube Formation--
Fig. 12
depicts tube formation in normal endothelial cells (Fig.
12A) and endothelial cells expressing rPAI-123
(Fig. 12B) after 14 days of growth. The tube formation is
extensive in the control cells and marginal in the transfected cells.
The rate at which the formation occurs is greatly reduced in the
rPAI-123-expressing cells. The BCECF AM probe is an
esterase substrate for cell viability and is used to determine the
ability of surviving cells to proliferate. Fig. 12 (C and
D) verifies the viability and proliferative capability of
both cells. However, it also shows the difference in the density of the
rPAI-123 (Fig. 12D) compared with the control
(Fig. 12C).
Proliferation--
Endothelial cells stably transfected with
rPAI-123 were tested for cell proliferation. The
differences in proliferation of endothelial cells transfected with
empty vector are compared with proliferation of
rPAI-123-transfected endothelial cells in Fig. 12E. During the initial 24-h growth period, the cells
secreting rPAI-123 grew at a slightly faster rate. However,
that difference was overtaken by the control endothelial cells in the
second 24-h period. The control cells continued to proliferate faster
than the rPAI-123-transfected cells with a 170% greater
rate of proliferation by the 96-h time point. All cells were maintained
in culture for an additional 10 days to determine whether the control
cells had reached plateau growth phase while the rPAI-123
transfectants continued to proliferate. At 336 h, the control
cells had increased in number by 288%, while the
rPAI-123-transfected cells had increased their number by
201%. The net result was greater than twice the number of control
cells when compared with the rPAI-123 cells at 336 h.
Migration--
The numbers of migrated endothelial cells
transfected with rPAI-123 is vastly reduced when compared
with the control cells that contain the empty vector (Fig.
12F). However, the numbers of migrating control cells
treated with VEGF increase 300%, and the
rPAI-123-transfected cell migration rate is reduced by one half. The effect of VEGF on migration of endothelial cells transfected with rPAI-123 is a 20-fold decrease when compared with the
empty vector control.
Angiostatin, a 38-kDa cleavage product of plasminogen, functions
as a regulator of angiogenesis by inhibiting endothelial cell
proliferation, migration, and tube formation (55, 56). It is known that
angiostatin is a cleavage product of plasminogen-containing kringle
domains 1-3 (57). However, the angiostatin-related mechanism(s) for
inhibition of endothelial cell proliferation, migration, and tube
formation remains unknown. Nor is it known which proteases convert
plasminogen to angiostatin (57).
The data that we have presented show that a truncated PAI-1 protein,
rPAI-123, mediates the formation of proteolytic
angiostatin-like protein fragments of an approximate 36- to 38-kDa
molecular mass and inhibits 80-kDa plasmin. The mechanism by which this
occurs is 2-fold. The first and seemingly preferred mechanism shown in Figs. 8 and 13 is due to
rPAI-123 cleavage of plasmin into 36- and 38-kDa fragments.
This takes place regardless of the permutation for combining
rPAI-123, uPA, and plasminogen. Increasing amounts of uPA
enhance the formation of angiostatin simply by producing more plasmin
for rPAI-123 to cleave. However, increasing amounts of
rPAI-123 inhibit the formation of proteolytic angiostatin
by reducing the amount of 80-kDa plasmin that is formed. This occurs in
a second mechanism where rPAI-123 binds either uPA (Fig. 9) or plasminogen (Fig. 10), thus reducing the number of uPA and
plasminogen interactions, which in turn reduces the amount of plasmin
that is produced (Figs. 9, 10, and
14). Even though the number of
rPAI-123 molecules exceeds that of uPA by 6- to 24-fold,
plasmin is still formed at approximately one-third the amount normally
formed in the absence of rPAI-123. This suggests that
plasminogen is able to dissociate uPA from rPAI-123, making
uPA available for binding plasminogen. Given sufficient quantities of
rPAI-123, increasing numbers of plasminogen become bound to
rPAI-123 and are unavailable for binding uPA. Likewise,
increasing amounts of rPAI-123 have an enhanced opportunity
to bind both uPA and plasminogen. The rPAI-123 protein
interaction with uPA and plasminogen inhibits the formation of the
normal 80-kDa plasmin and the formation of the 36- to 38-kDa
proteolytic angiostatin-like fragments. These data strongly suggest
that rPAI-123 exposes binding sites for uPA, plasminogen,
and plasmin that provide the truncated inhibitor with two pathways for
regulating plasmin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3 integrins
(20, 21), plasmin, and recycled uPAR. The regulatory mechanisms of
PAI-1 are also regulated by other molecules in the proteolytic pathway.
PAI-1 and plasmin both have vitronectin binding sites. The serine
protease activity of plasmin degrades Vn such that one of the PAI-1
binding sites is mostly lost. This results in destabilization of the
Vn·PAI-1 complex and a loss of PAI-1 function as an inhibitor of
plasmin formation through the plasminogen activator pathway. This same
PAI-1 binding site on Vn overlaps a heparin binding site. The protease
activity of plasmin completely abolishes the Vn binding site for
heparin and exposes a plasminogen binding site (9). Heparin interaction
with PAI-1 increases the specificity of PAI-1 for thrombin (22).
Variations in Vn conformational states can convert PAI-1 inhibitory
specificity from the plasminogen activators to thrombin (23, 24).
-sheet to exhibit inhibitory activity (35-37). Declerck et al. (38,
39) showed that the reactive loop can be cleaved by uPA between
residues 346 and 347, turning PAI-1 into a substrate. In the inactive
configuration of PAI-1 (not bound to vitronectin), the reactive loop is
spontaneously inserted into the folds of PAI-1 at physiological
temperatures (29, 40). This conformation is stable due to the loop
insertion into strand 4a of the PAI-1 structure (41). A Lys-323 residue located within the
-strand s5A is thought to play an important role
in the regulation of the latent/inactive state. This residue forms a
hydrogen bond with Asn-150 (or 144 in mature protein) on the s3A/helix
F loop of PAI-1 (42).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Truncated rPAI-123 gene relative
to full-length human and porcine PAI-1. This drawing depicts the
rPAI-123 gene relative to the human and porcine PAI-1
genes. It also illustrates the PAI-1 gene sequences/regions that code
for amino acids known to be important binding sites for various
regulatory proteins in the proteolytic and fibrinolytic pathways.
A (Invitrogen, Carlsbad, CA). The TOP 10 strain of Escherichia coli was transformed by
electroporation of the ligation reaction in 2-mm cuvettes containing 50 ng of the ligation reaction/100 µl of electroporation competent TOP
10 cells using a BTX Electro Cell Manipulator 600 (BTX, San Diego, CA).
The electroporation conditions were 2.5-kV resistance, 129 ohm
and a charging voltage of 2.45 kV. The pulsed cells were incubated at
37 °C for 1 h and then transferred to an agar plate containing
low salt Luria broth and 50 µg/ml zeocin. Following an overnight
incubation at 37 °C, colonies were selected and grown in low salt LB
broth for 5-7 h at 37 °C. The DNA from each colony growth was
isolated on a Qiagen miniprep spin column (Qiagen, Inc., Valencia, CA).
The purified DNA from each expanded colony was double-digested to verify the presence of the gene insert. Positive isolates from the
restriction enzyme digests (Fig. 2) were
verified by sequencing.
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Fig. 2.
Isolation and cloning of porcine PAI-1 gene
fragments. The agarose gel shows the results of cloning fragments
rPAI-114 and rPAI-123 into pGAPZ A yeast
expression shuttle vector. The PCR-amplified PAI-1 gene fragments were
1137 and 555 bp, respectively. The PCR-amplified
rPAI-114 and rPAI-123 are shown adjacent to
their respective vector-amplified fragments that were restriction
enzyme-digested from pGAPZ
A with EcoRI and
XbaI. Lane 1, DNA molecular weight marker;
lane 2, supercoiled pGAPZ
A containing the 1137-bp PAI-1
DNA fragment; lane 3, EcoRI and XbaI
enzyme digest of pGAPZ
A containing the 1137-bp PAI-1 DNA fragment;
lane 4, reverse transcription-PCR-isolated/amplified 1137-bp
PAI-1 DNA fragment; lane 5, supercoiled pGAPZ
A containing
the 555-bp PAI-1 DNA fragment; lane 6, EcoRI and
XbaI enzyme digest of pGAPZ
A containing the 555-bp PAI-1
DNA fragment; lane 7, reverse
transcription-PCR-isolated/amplified 555-bp PAI-1 DNA fragment.
A glyceraldehyde-3-phosphate dehydrogenase promoter. The linear DNA was transfected into
electroporation competent P. pastoris strain of yeast
according to the manufacturer's protocol (Invitrogen). The transfected
yeast cells were streaked onto YPD/zeocin (100 µg/ml) plates.
Resulting clones were streaked onto YPD plates a second time to ensure
integration of the transfected DNA into the yeast genome. Individual
clones were selected after 2-3 days of growth on the YPD plates and
grown in YPD medium at 30 °C for 1 day. The transfected cells were
pelleted, and the supernatant containing the secreted recombinant PAI-1
protein was collected and verified on a silver-stained 4-20% gradient SDS-polyacrylamide gel (Fig. 3).
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Fig. 3.
rPAI-123 protein expression and
isolation. A single colony of rPAI-123-transfected
P. pastoris yeast cells was grown in YPD medium at 30 °C.
The supernatant containing the secreted protein was collected,
concentrated, and expanded after the first day of growth. Likewise, on
day 2 the secreted protein was collected and concentrated from a larger
culture volume. The isolated rPAI-123 protein corresponds
to the appropriate molecular mass of 18 kDa (lane 5).
Lane 1, protein molecular weight marker; lane 2,
P. pastoris-secreted protein, day 1; lane 3,
P. pastoris-secreted protein, day 2; lane 4,
rPAI-123 protein, day 1; lane 5,
rPAI-123 protein, day 2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 4.
Chromozym assay to detect plasmin
formation. The Chromozym PL used in the assay measures the
formation of plasmin. The truncated rPAI-123 protein
(lane 5) inhibited ~50% of the plasmin formed by the
activity of uPA as compared with the control containing zero units of
hPAI-1. The rPAI-123 protein lacks the reactive center loop
required for activation of plasminogen. Lane 1, P. pastoris yeast cells; lane 2, huPAI-1 (132.0 IU);
lane 3, huPAI-1 (66.0 IU); lane 4, huPAI-1 (0 IU); lane 5, rPAI-123; lane 6,
rPAI-114.
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Fig. 5.
Zymography functional assay. The
zymography technique allows visualization of plasmin formation. The
proteolytic protein near 36 kDa that is formed as a result of
rPAI-123 interaction with either plasminogen or plasminogen
and uPA is clearly visible in lanes 6, 7,
10, and 11. The enhancement of the proteolysis is
seen in lanes 10 and 11. Inhibition with
increasing amounts of rPAI-123 is seen in lanes
8 and 12. Full-length recombinant PAI-1 is named
rPAI-114. An rPAI-123 protein with a reactive center loop
at the carboxyl terminal is named rPAI-124. In the
following description of the lanes, 14 = rPAI-114, 24 = rPAI-124, and 23 = rPAI-123: Lane 1, 24 (50 nM) + Plg;
lane 2, 24 (50 nM) + uPA + Plg; lane
3, 14 (50 nM) + Plg; lane 4, 14 (50 nM) + uPA + Plg; lane 5, 23 (50 nM);
lane 6, 23 (30 nM) + Plg; lane 7, 23 (50 nM) + Plg; lane 8, 23 (120 nM) + Plg; lane 9, 23 (30 nM) + uPA; lane
10, 23 (30 nM) + uPA + Plg; lane 11, 23 (50 nM) + uPA + Plg; lane 12, 23 (120 nM) + uPA + Plg; lane 13, Plg; lane
14, hPAI-1(1.0 µg) +uPA + Plg; lane 15, molecular
mass marker.
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Fig. 6.
Zymography analysis of the effect of
vitronectin on rPAI-123 activity. Zymography
techniques were used to analyze the effect of vitronectin (Vn) and
various components of the PAI-1 activation buffer on the stability and
functionality of rPAI-123. Lanes 9-12 represent
30 nM rPAI-123 reacted with uPA and plasminogen
in the presence of Vn and the buffer components. None of the tested
variables altered the formation of the resulting 36-kDa proteolytic
protein. In the following description of the lanes, 23 = rPAI-123: Lane 1, 23 + Plg + Tween 20/TBS;
lane 2, 23 +Plg + BSA/TBS; lane 3, 23 +Plg + Tween 20/TBS/BSA; lane 4, 23 + Plg + TBS; lane 5,
23-Vn +Plg + Tween 20/TBS; lane 6, 23-Vn + Plg + BSA/TBS;
lane 7, 23-Vn + Plg + Tween 20/TBS/BSA; lane 8,
23-Vn + Plg + TBS; lane 9, 23-Vn + uPA + Plg + Tween 20/TBS;
lane 10, 23-Vn + uPA + Plg + BSA/TBS; lane 11,
23-Vn + uPA + Plg + Tween 20/TBS/BSA; lane 12, 23-Vn + uPA + Plg + TBS; lane 13, uPA + Plg + Tween 20/TBS; lane
14, uPA + Plg + BSA/TBS; lane 15, uPA + Plg + Tween
20/TBS/BSA; lane 16, uPA + Plg + TBS; lane 17, Vn + uPA + Plg + Tween 20/TBS; lane 18, Vn + uPA + Plg + BSA/TBS; lane 19, Vn + uPA + Plg + Tween 20/TBS/BSA;
lane 20, Vn + uPA + Plg + TBS.
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Fig. 7.
Western blot probed with anti-kringles 1-3
(angiostatin). Samples from the zymogram in Fig. 5 were
electrophoresed on a 4-20% SDS, nonreducing polyacrylamide gel. The
analysis is of the proteins formed in the interactions of
rPAI-123 with plasminogen (Plg) and uPA. The samples were
transferred to a nitrocellulose membrane and probed for angiostatin
(kringles 1-3 of plasminogen). Lane 1, broad range protein
molecular mass marker; lane 2, rPAI-124 protein + Plg; lane 3, rPAI-114 protein + uPA + Plg;
lane 4, rPAI-124 protein + Plg; lane
5, rPAI-114 protein + uPA + Plg; lane 6,
rPAI-123 (50 nM); lane 7,
rPAI-123 (30 nM) + uPA; lane 8,
rPAI-123 (30 nM) + Plg; lane 9,
rPAI-123 (120 nM) + Plg; lane 10,
rPAI-123 (30 nM) + uPA + Plg; lane
11, rPAI-123 (120 nM) + uPA + Plg.
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Fig. 8.
Varied permutations for rPAI-123,
uPA, and plasminogen interactions. The mechanism by which
rPAI-123 induces angiostatin was analyzed by either: 1)
reacting uPA and rPAI-123 for 1 h at room temperature
(RT) and then adding Plg for an additional 1-h incubation at 37 °C
or 2) reacting uPA and Plg at 37 °C (plasmin) before adding 30 mM rPAI-123 for an additional 1 h at
37 °C. In the following description of the lanes, 23 = rPAI-123, 24 = rPAI-124, and RT = room temperature. Lane 1, uPA (10 nM) + Plg,
1 h 37 °C; lane 2, uPA (40 nM) + Plg,
1 h, 37 °C; Lane 3, uPA (10 nM) +Plg,
1 h, 37 °C + 23, 1 h, 37 °C; lane 4, uPA (40 nM) + Plg, 1 h, 37 °C + 23, 1 h, 37 °C;
lane 5, uPA (10 nM) + 23, 1 h, RT + Plg,
1 h, 37 °C; lane 6, uPA (40 nM) + 23, 1 h, RT + Plg, 1 h, 37 °C; lane 7, molecular
mass marker.
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Fig. 9.
Binding of rPAI-123 to uPA.
The effects of rPAI-123 on uPA formation of plasmin were
tested by adding either 30 or 120 nM rPAI-123
to 5 nM uPA either before or after an incubation with 33 nM Plg. To ensure that all Plg or uPA was exhausted, a
second amount of either one was added for an additional hour at
37 °C. In the following description of the lanes, 23 = rPAI-123: Lane 1, uPA + Plg; lane 2,
23 (30 nM) + uPA, then Plg; lane 3, uPA + Plg,
then 23 (30 nM); lane 4, 23 (120 nM) + uPA, then Plg; lane 5, 23 (120 nM) + uPA, then
Plg, second Plg; lane 6, uPA + Plg, then 23 (120 nM); lane 7, uPA + Plg, then 23 (120 nM), second Plg; lane 8, 23 (120 nM)+ uPA, then Plg; lane 9, 23 (120 nM) + uPA, then Plg, second uPA; lane 10, uPA + Plg, then 23 (120 nM); lane 11, uPA + Plg, then
23 (120 nM), second uPA; lane 12, molecular mass
marker.
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Fig. 10.
Binding of rPAI-123 to
plasminogen. The affinity of rPAI-123 for plasminogen
was tested in varied permutations of the order in which reactants were
added. Plasminogen was held constant at 33 nM while the
amounts of rPAI-123 and uPA were varied. In the following
description of the lanes, 23 = rPAI-123: Lane
1, Plg; lane 2, uPA (5 nM) + Plg;
lane 3, uPA (5 nM) + Plg, second Plg; lane
4, uPA (0.5 nM) + Plg; lane 5, uPA (0.5 nM) + Plg, second Plg; lane 6, 23 (30 nM) + Plg, then uPA (0.5 nM); lane
7, 23 (30 nM) + Plg, then uPA (5 nM),
second Plg; lane 8, 23 (60 nM) + Plg, then uPA
(5 nM) + Plg; lane 9, 23 (60 nM) + Plg, then uPA (5 nM), second Plg; lane 10, 23 (60 nM) + 0.001 uPA + Plg; lane 11, 23 (30 nM) + Plg, then uPA (0.5 nM); lane
12, 23 (30 nM) + Plg, then uPA (0.5 nM),
second Plg; lane 13, 23 (60 nM) + Plg, then uPA
(0.5 nM); lane 14, 23 (60 nM) + Plg,
then uPA (0.5 nM), second Plg; lane 15,
molecular mass marker.
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Fig. 11.
Endothelial cell incubation with exogenous
rPAI-123. The function of angiostatin is to inhibit
endothelial cell proliferation, migration, and tube formation. The
series of experiments in A-E were performed to do an
initial observation of exogenous rPAI-123 effects on
endothelial cell growth, attachment/detachment, and tube formation.
After 5 days in culture, the cells treated with exogenous
rPAI-123 displayed obvious differences in proliferation,
morphology, tube formation, and detachment. A and
B, tube formation. A represents the untreated
control cells displaying confluency and tube formation; B
shows rPAI-123-treated endothelial cells. Their rate of
proliferation is reduced such that they did not reach confluency by day
5 and, therefore, did not display tube formation. C,
apoptosis was evaluated after observing detachment of endothelial cells
treated with rPAI-123. The results of the annexin V binding
assay showed that 65% of the combined rPAI-123 detached
and adherent cells were undergoing apoptosis, whereas only 15% of the
detached and adherent control cells were apoptotic. This effect was not
detectable in smooth muscle cells. D, proliferation was
determined by counting the cells in each test sample at various time
points. The results were verified by performing a CellTiter 96 Aqueous
One Solution cell proliferation assay. The rate of proliferation of
rPAI-123-treated cells was reduced ~30% in 96 h as
compared with control endothelial cells that were untreated or to which
yeast protein was added to culture medium (D).
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Fig. 12.
The effect of stably transfected
rPAI-123 on endothelial cells. The DNA containing the
sequences coding for rPAI-123 protein was ligated into
pCMV/Myc/ER vector to enable synthesis of the protein in the
endoplasmic reticulum and secretion of the protein. The vector
containing rPAI-123 was electroporated into endothelial
cells. The expression of rPAI-123 was verified in a
Chromozym assay. The effects of the secreted protein on the endothelial
cells was examined. A-D, tube formation. The images depict
tube formation in normal (empty vector) endothelial cells
(A) and endothelial cells expressing rPAI-123
(B) after 14 days of growth. The tube formation is extensive
in the control (empty vector) cells and vastly reduced in the
rPAI-123-transfected cells. The BCECF AM probe is an
esterase substrate for cell viability and is used to determine the
ability of surviving cells to proliferate. C and
D show endothelial control cells and
rPAI-123-transfected endothelial cells to which the BCECF
AM substrate has been added to verify the viability and proliferative
capability of both cells. This shows the difference in the density of
the rPAI-123 (D) compared with the control
(C). E, proliferation. The proliferation of
rPAI-123-transfected cells is compared with control (empty
vector) endothelial cells in E. Cells were plated at an
equal density, collected, and counted on the designated time points.
There is a continuous increase in proliferation of the endothelial
cells such that at the end of 336 days there are twice the number of
rPAI-123-transfected cells at 336 h. F,
migration. The migration of endothelial cells transfected with
rPAI-123 or empty vector was measured in a Boyden chamber
after a 4.5-h incubation in serum-free DMEM containing either VEGF or
no growth factors. The migrated cells were fixed in 4% buffered
formalin, stained with toluidine blue, and mounted on glass slides.
Five fields from each of three test samples were counted at × 200 magnification.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 13.
The mechanism by which rPAI-123
inhibits plasmin and induces angiostatin. Varied permutations for
rPAI-123, uPA, and Plg interactions were tested to
determine whether the order in which 30-60 nM
rPAI-123, uPA, and Plg were added to a reaction mixture
altered the appearance of the proteolytic angiostatin-like product.
Regardless of the order, 30-60 nM of rPAI-123
is able to cleave plasmin. The cleavage product is proteolytic
angiostatin. Increasing the amount of uPA in the reaction enhances the
formation of angiostatin by providing more plasmin for
rPAI-123 to cleave. The affinity of uPA for plasminogen is
greater than for rPAI-123, because either permutation
results in comparable amounts of angiostatin.
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Fig. 14.
Inhibition of angiostatin by
rPAI-123. The truncated PAI-1 protein,
rPAI-123, has a binding affinity for both uPA and
plasminogen. Plasminogen can dissociate uPA from rPAI-123.
The affinity of uPA for plasminogen enables plasmin to be formed at a
reduced level. Increasing molecules of rPAI-123 reduce the
potential of uPA and plasminogen interactions.
When rPAI-123 protein was added exogenously to endothelial cells, proliferation was decreased by ~30% in the initial 48 h and tube formation was inhibited. Endothelial cells transfected with rPAI-123 contained within pCMV/Myc/ER vector displayed the same profile of reduced proliferation and tube formation. Moreover, the rPAI-123-transfected cells show a 20-fold reduction in migration in the presence of VEGF when compared with the control cells. Additionally, we demonstrate endothelial cell specificity in the apoptosis assay where we determined that 47% of the rPAI-123-treated endothelial cells underwent apoptosis within the first 24-48 h, while only 12% of the rPAI-123-treated smooth muscle cells were apoptotic. The 36-kDa proteolytic fragment that we have identified exhibits those properties inherent to angiostatin.
The significant apoptotic index in rPAI-123-treated cells provides a potential explanation for the reduction in proliferation of rPAI-123-treated and -expressing endothelial cells. The reduction in proliferation in turn reduces the tube formation, because the tubes do not begin to form until endothelial cells reach quiescence/confluence. The apoptotic and proliferation results that we have documented are in concurrence with results reported by Claesson-Welsh et al. (58).
The reactive loop containing the uPA binding site needed for inactivation of uPA was deleted from rPAI-123, thus releasing the strained loop conformation as well as the 59 amino acids on the amino terminus containing important binding sites. By all definitions of "active" PAI-1, rPAI-123 would not be expected to have activity. However, we have shown that this truncated protein is capable of inhibiting the 80-kDa plasmin by two mechanisms. First, it cleaves plasmin to produce angiostatin-like proteolytic proteins. The proteolytic activity of the 34-kDa angiostatin is ultimately inhibited by increasing molecules of rPAI-123 that are available for binding uPA and/or plasminogen. In this second mechanism, rPAI-123 reduces the numbers of uPA·Plg interactions, thus reducing the amount of plasmin production. This truncated PAI-1 conformation appears to be exposing sites that participate in a functional role for PAI-1 in generating angiostatin fragments from plasmin. It has been suggested that, in the transition between latent and active states, such sites may become available for interaction with other regulatory proteins involved with proteolysis, fibrinolysis, adhesion, or angiogenesis (48, 58-60).
There are reports that metalloproteinases are responsible (61-65) for
cleavage of plasminogen into angiostatin. In some reports, it has been
demonstrated that neoplastic cells produce an enzyme that converts
plasminogen to angiostatin (66, 67). In other findings,
metalloproteinases expressed by tumor-associated macrophages produce
angiostatin fragments in tumor cells (62). Falcone et al.
(68) recently reported that angiostatin fragments (~36 kDa and 48 kDa) are formed as "by-products" of membrane-bound plasmin regulation in normal cells in the absence of metalloproteinases. They
also showed that the <36-kDa fragment has enzymatic activity but does
not bind to the cell surface. The larger fragment also has enzymatic
activity and is able to inhibit endothelial cell proliferation (68).
There are reports that urokinase and sulfhydryl donors are responsible
for the conversion of plasminogen into angiostatin (69). A reductase
secreted by fibrosarcoma and Chinese hamster ovary cells reduced
plasmin disulfide bonds to generate angiostatin (67, 70). O'Mahony
et al. (71) have shown that transforming growth factor-1
inhibits angiostatin from forming in human pancreatic cancer cells. The
addition of exogenous PAI-1 inhibited the cleavage of plasminogen into
angiostatin (71). Collectively, these data suggest that cleavage of
angiostatin from plasminogen occurs by more than one mechanism. We show
a role for PAI-1 in the induction of proteolytic angiostatin from plasmin. We are pursuing the in vivo events that could
result in a PAI-1 conformation having the function we have
demonstrated, because this would be an important finding relative to
tumorigenesis and vascular disease.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the editing and graphic expertise of Luanna R. Bartholomew, Ph.D.
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FOOTNOTES |
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* This work was supported by Research Grant R01-HL59590 from the NHLBI, National Institutes of Health, and by a Pacific Vascular Research Foundation Award.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 should be addressed: Dept. of Surgery,
Vascular Surgery, Borwell 530 E, 1 Medical Center Dr., Dartmouth Medical School, Lebanon, NH 03756. Tel.: 603-650-8597; Fax:
603-650-4928; E-mail: mary.j.mulligan-kehoe@dartmouth.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M006434200
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; uPA, urokinase plasminogen activator; tPA, tissue plasminogen activator; uPAR, uPA receptor; PAI-1/-2, plasminogen activator inhibitors 1 and 2; huPAI-1, human PAI-1; poPAI-1, porcine PAI-1; rPAI-1, recombinant PAI-1; Vn, vitronectin; VEGF, vascular endothelial growth factor; bp, base pair(s); PCR, polymerase chain reaction; YPD, Yeast Extract Peptone Dextrose medium; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; FCS, fetal calf serum; BCECF AM, 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester; HBSS, Hanks' balanced salt solution; Plg, plasminogen.
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