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Cell-Surface Plasminogen Activation Causesa Retraction of In Vitro Cultured
Human Umbilical Vein Endothelial Cell Monolayer
By Grazia Conforti, Carmen Dominguez-Jimenez, Ebbe Rsnne, Gunilla Hsyer-Hansen, and Elisabetta Dejana
Vascular endothelium formsa dynamic interface between
blood and underlying tissues. Endothelial monolayer
integrity is required for controlled vascular permeability and to
preclude exposureof subendothelial cellmatrix to circulating cells. Recent studies
have established
that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen
activator (uPA).In the present study,we provide evidence
that in EC, uPA receptor is present
in focal contacts and
at
cell-cell contact sites. In these cells, addition of plg and
uPA to confluent EC generates a retraction of the monolayer that is evidenced byloss of cell-cell contacts and increase in monolayer permeability. The phenomenon is re-
versible evenafter 6 hoursof plg-uPA treatment. Inhibition
of plg-uPA effect is obtained with plasmin inhibitors, as
well as reagents
that blodc binding of uPA plg
or to the cell
surface. The retractive effectof plg-uPA is concomitant
to
surface activation
of plasminogen and
to the lossof cell-cell
and cell-matrix contacts.
We concludethat the cell-surface
activation of plg can induce EC retraction, possibiy by causing proteolysisat specific cell-cell contacts and cell-matrix
sites. This process may
be important in mediating
the passage of metastatic tumor cells through
an intact EC monolayer aswell as in regulating contactsbetween circulating
cells and endothelium.
0 1994 by TheAmerican Society of Hematology.
E
this receptor has a very
high
The cloning and
purification of uPAR have shown that it is a highly glycosylated polypeptide with an M, of 55,000 to 60,000,20which is
linked to theplasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor.2'uPAR can bind both the
active two-chain uPA and the proenzyme single-chain prou P A . ~Binding
~
of both forms is mediated by the NH2-termina1 growth factor domain of uPA contained in the 16-kD
A
Thus,
the bound enzyme is catalytically active,
and bound pro-uPA can be activated by plrn?' Besides localizing plg activation to the cell surface, the presence of
these receptors results in strong acceleration of the rate of
activation of pro-uPA to uPA by plm13925
and possibly in
direct activation of pro-uPA.26 The cell-surface location
protects plm from its physiologic inhibitor a2-antiplm.'3327328
These events constitute important regulatory
mechanisms at sites of pericellular proteolysis.
Most malignant cells have been described to overproduce
plg a ~ t i v a t o r sIn
. ~ ~addition,they may display autocrine saturation of uPAR with pro-uPA or active U P A . ~
In this report, we have investigated whether the uPAuPAR system could intervene in the EC retraction. To this
goal, we used human umbilical vein ECs, as they have been
shown to have uPAR, to produce plg-activator inhibitor
type 1 (PAI-1)'5,'6,3'.32
and tohave plg binding
NDOTHELIAL CELLS (ECs) play a very important
role in mediating the passage to tissues of normal and
malignant tumor cells. In fact, the first interaction between
circulating malignant cells and their target organ occurs
when they reach local capillary endothelium. Upon adhesion to ECs, malignant cells cause a retraction of the ECs
that uncovers the underlying basement membrane, which
becomes directly accessible for digestion and invasion.'
Therefore, retraction of the ECs may be an important step
in the extravasation of blood cells and of malignant (metastatic) cells.
Retraction must require loosening or destruction of some
cell-cell and cell-basement membrane connections, a process that might require proteases that must be active on the
surface of invading-crossing cells. The most thoroughly
known cell-surface proteolytic system is the plasminogen
(plg) cascade regulated by the urokinase-like plasminogen
activator (uPA) and its receptor (uPAR).~This system has
been shown to be directly involved in several steps of tumor
cell invasion3-'' and inthe inflammatory response'' because
it provides the cell surface with regulated and localized plasuPAR has been identified
min (plm) a ~ t i v i t y . ' ~A, 'specific
~
and cloned14and shown to be present on E C S ' ~ along
, ' ~ with
specific binding sites for plg/plm.17.18The plg binding site is
characterized by relatively low affinity and high capacity,
The
and it recognizes the lysine binding-sites of ~ l g . " , ' ~
number of uPAR molecules present on cells is lower, but
From the Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; and Finsen Laboratory, Copenhagen, Denmark.
Submitted April 12, 1993; accepted October 4, 1993.
Supported by Associazioge Italiana per la Ricerca sul Cancro, the
Italian National Research Council (CNR) Special Projects ("Biotecnologie e Biostrumentazione" and Applicazioni Cliniche della Ricerca Oncologica),and the North Atlantic Treaty Organization (Collaborative Research Grant No. 920010).
Address reprint requests to Grazia Conforti, MD, Istituto di Ricerche FarmacologicheMario Negri, Via Eritrea 62,2015 7Milan0,
Italy.
The publication COSIS of this article were defrayed in part by page
charge payment.This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate thisfact.
01994 by The American Society of Hematology.
0006-4971/94/8304-0027$3.00/0
994
MATERIALS ANDMETHODS
Materials. The following reagents were used and are listed below withtheir source: human glu-plg (20.2CU/mg) and human plm
(15.7 CU/mg) (Kabi Vitrum, Stockholm, Sweden); human urokinase (50-kD molecular weight, 100,000 IU/mg) (Persolv Richter)
was a kind gift from LepetitSPA (Milano, Italy); human prourokinase was a kind gift from Farmitalia-Carlo Erba Srl(Nerviano, Italy);t-PA (792,000 IU/mg and 704,225 IU/mg) (BiopoolAB,
Umei, Sweden); low molecular weight urokinase (33 kD,160,000
IU/mg) (AmericanDiagnostica Inc, Greenwich, CT); trasylol
(Bayer, Leverkusen,Germany); recombinant human amino terminal fragment (rhATF) of uPA was purified from LB6-Cl AF cellconditioned medium"; human plasma vitronectin (vn) and fibronectin (fn) were prepared as previously described," mouselaminin
purified from the murine tumor EHS, was a kind gift from Dr G.
Taraboletti (Istituto di Ricerche Farmacologiche Mario Negri,
Bergamo, Italy), c-aminocaproic acid (cACA), paraformaldehyde, sucrose,tween-80(tw-80) (MERCK-Schuchardt, Darmstadt, Germany), crystal violet, fluorescein-tagged phalloidin from Amanita
phalloides, bovineand human serum albumin (BSA and HSA), and
Blood, Vol83, No 4 (February 15). 1994: pp 994-1005
PLASMINOGEN ACTIVATION ONECSURFACE
a2-anti-plm (Sigma Chemical CO,St Louis, MO); diff-quik(Men
Dade AG, Dudingen, Switzerland);mowiol4-88
(Hoechst,
Frankfurt/Main, Germany); iodogen (Pierce Chemical CO,Rockford, IL); transwells tissue
culture dishes (0.4-pmpore size, polycarbonate membrane filter)(Costar, Cambridge, MA); I4C-methylated
protein molecular-weight standards (Mr200,000myosin,Mr
100,000 to 92,500 phosphorylase b, Mr 69,000 BSA, Mr 46,000
ovalbumin, Mr 30,000 carbonic anhydrase, Mr 14,3000 lysozyme)
(Amersham International, Buckinghamshire, UK); phosphatidylinositol-specific phospholipase C fromBacilluscereus(PI-PLC)
(Boehringer Mannheim GmbH, Mannheim, Germany); camerfree Na12’I (NewEnglandNuclearResearch
Products, Boston,
MA); and all culture reagents (GIBCO, Paisley, UK).All other reagents were ofthe highest chemical grade.
Antibodies. Anti-P3subunit rabbit serum has been de~cribed,~’
antiplatelet EC-adhesion-molecule (PECAM) rabbit IgG36 was a
kind gift from Dr P. Newman (Blood Research Institute, Milwaukee, WI), antihuman-fn rabbit serum was a kind gift from Dr G.
Marguerie (Institut National de la Santi et de la Recherche Medicale, Grenoble Cedex, France), antimouse laminin rabbit serum
was a kind gift from Dr G. Taraboletti (Istituto di RicercheFarmacologiche Mario Negri, Bergamo, Italy);
anti-uPAR monoclonal
antibody (MoAb) clone R4 (IgG) has been described3’; anti-intercellular adhesionmolecule-l (ICAM-I) MoAb (~upernatant)~~
was
a kind gift fromDr N. H o g (Imperial Cancer ResearchFund, London, UK). Secondary antibodies used in immunofluorescence experiments were from Dakopatts (Glostrup, Denmark).
Cell cultures. ECswere isolatedfrom normal-term umbilical
cord veins by collagenase perfusionas previously de~cribed.’~
Cells
were grown on tissue culture dishes coated with gelatin (0.5%)in
20% newborn calf-serum/Mvl99 medium supplemented with EC
growth supplement (ECGS) (prepared from bovine brain) (50 pg/
mL), heparin ( 100 pg/mL), and penicillin (100 U/mL)/streptomycin (100pg/mL) (pen/strep) and used betweenthe first and the third
passage.
Human saphenous-vein ECs were kindly provided by Dr C. de
Castellarnau (Fundacio d’lnvestigacioSant Pau, Barcelona, Spain).
Cells were isolated from specimens of normal veins as previously
described.34 Cells werecultured in M199,20% human serum supplemented with ECGS (50
pg/mL), heparin (100 pg/mL), and pen/
strep, on 0.5%gelatin coating and used at the fourth passage.
Immunofluorescence. EC were plated on glass coverslips coated
with 0.5% gelatin. They were incubated with the primary specific
antibodies before or after being fixed. uPARstaining on living cells
was previously describedby Pollanen et al.39Cells were fixed with
( 1 5 min3% paraformaldehyde in phosphate-buffered saline (PBS)
utes at room temperature) containing 2% sucrose and washed three
times with PBS. They were then incubated with the appropriated
secondary antibody-fluorochromeand conjugated for 45 minutes
at 37°C in PBS-I%BSA. After this step, some samples were permeabilized by incubating with0.5%Tx-100 in HEPESbuffer (20
mmol/L HEPES, 300mmol/L sucrose, 50mmol/L NaCI, 3 mmol/
L M&) for 4 minutes at 4°C. After extensive washing
(three times
with PBSand once with PBS-O.1% BSA), for double-labeling, samples were incubated with the second specificantibody followed by
the second fluorochrome-conjugatedantirabbit or antimouse antibodies, which werediluted 1:40 in PBS- 1% BSA for 45 minutes at
37’C. Samples not permeabilized before were permeabilized now.
Whencell-surface proteins had to be detected (such as uPAR,
PECAM, or vn receptor) the permeabilization step was performed
only at the end of the immunostaining protocol to avoid any nonspecific binding of the antibodies inside the cells. Inthe case of vinculin, cells were permeabilized beforethe addition of antivinculin
antibodies. Labeling withor without permeabilizationwas compabe observed with permeabilrable, exceptthat a clearer image could
ized cells. After extensive rinsing, coverslips mounted
were
in mow-
+
995
io1 4-88. Routine observations were performedin a Zeiss Axiophot
photomicroscope equipped for epifluorescence (Carl &is, Oberkochen, Germany) and fluorescent images
were recorded on Kodak
400 films (Eastman-Kodak, Rochester, NY). Incubation with PIPLC (0.5 U/mL) was performed in serum-free M199for 1 hour at
37°C before cell
incubation with anti-uPAR MoAbs.
Light microscopy analysis of EC-monolayer retraction. ECs
were grown to confluency in 96- or 24-well culture dishes coated
with 0.5% gelatinand proteolytic treatment was performed on EC
monolayers previously maintained in serum-free M199 (50 minutes at 37°C). Retraction was studied by the addition of plg in fresh
serum-free M 199, followed after
IO minutes by the addition of uPA.
In some samples, ATF, tACA, and a,-anti-plm were also present
and were added together with plg. Morphologic changes of
the
monolayer were observed at different times by light microscopy.
Samples were photographed in a Zeiss IM 35 photomicroscope
(Carl Zeiss) on Kodak plus-Xpan 125 films (Eastman-Kodak).
In preliminary experiments,we partially definedthe composition
of the extracellular EC matrix. ECs seeded on 0.5% gelatin-coated
coverslips,were grown for7 days to reach the confluence in serumcontaining medium. By indirect-immunofluorescencemicroscopy
using anti-fn and antilaminin antibodies, we found that a large
amount of laminin and fn was present on the EC matrix after 7
days of culture (not shown). Proteoglycans have been previously
described as a component of the EC matrix by Mertens et aLm
Radioisotope labeling of proteins. plg and HSA were radioiodinated with camer-free Na12’I (7 to 1 1 pCi/pg ofprotein) using the
Iodogen method for 15 minutes at room temperature according to
the manufacturer’s procedure. The reaction was stopped by excess
K1 and 1251-labeled
proteins separated from free 1251 on a PD-IO
column (Pharmacia LKB Biotechnology, Uppsala, Sweden).
Specific activityof the proteins was 3 X IO6 cpm/pg for plg and
IO6 cpm/pg for HSA.I2’I-HSAwas dialyzed before each experiment.
12SI-albuminendothelial permeabilityassay. To quantify the
effect of the proteolytic treatment on EC-monolayer permeability
we measured the passage of I2’I-HSAthrough EC confluent monolayersusing the Transwell system.4143Cellswereplated on fncoated polycarbonate filters (2 &well) placed on the upper compartment of the transwell chamber. After 7 days of culture, cells
were kept for 50 minutes at 37°C in serum-free M199, which was
then replaced with 100 pL serum-free M199 containing ”’I-HSA
( IO6 cpm) and 0.54 pmol/L plg in the upper compartment. In the
lower compartment, 600 pLof serum-free M 199 waspresent. Proteolysis was started by the addition of uPA to the upper compartment as described above. In some experiments, ATF, cACA, a2anti-plm, and trasylol were presentduring the incubation and were
added together withplg and I2’I-HSA. Atdifferent time points, 100p L samples were withdrawn from the lower compartment (replaced
with serum-free M 199) and counted in a y counter. At the end of
the experiment, monolayers on the filterswere diffquik fixed, crystal-violet stained, and photographed as described above.The discontinuities in ECmonolayer generatedby the plg-uPA treatment were
quite different in number and size within replicates. However,the
values of replicates ofuntreated monolayers were verysimilar. The
inset of the figures shown with this kind of experiment have been
added to partially overcomethis intrinsic problem.
Statistical analysis. The Student’s t-test was used to determine
if there were significant differences betweenexperimental groups.
Samples fromat least three independent experimentswere grouped
for this statistical analysis. Probability valuesof .05 were required
for statistical significance.
EC-mediated plg cleavage by uPA. ECs were grown to confluency in 96-well culture dishes coated with 0.5% gelatin. After serum-free M199 incubation (50 minutes 37’C), 0.8 pmol/L unlabeled plg containing ‘”I-plg ( I .5 X IO6 cpm/well) was added and
996
CONFORTI ET AL
cells incubated for 10 minutes at 37'C. In some wells, after this inwhere it codistributed with PECAM (Fig 2, compare A with
cubation, theradiolabeled plg mixture was substitutedwith 0.8
B). Nonimmune rabbit serum in double label with uPAR
pmol/L unlabeled plg, corresponding to 200-fold molar excess of
gave a negative staining (Fig 2D). When an anti-ICAM-l
the cell-bound '2sI-plg,as calculated by counting in a y counter the
MoAb was used as control, a bright diffuse staining of the
unbound-radiolabeled plg, removed from the monolayer after 10
cell surface was observed with no specific localization of the
minutes of incubation withcells. uPA (50 nmol/L) was then added
antibody at cell-cell contacts (Fig 2F). Furthermore, suband cells incubated for a further I5 minutes at 37°C. At this time,
confluent
ECs also showed a more marked uPAR staining
trasylol (800 KIU/mL) was added in the well, supernatants were
at the cell border (Fig 2E), thus excluding a nonspecific acremovedandputinavessel
containing electrophoresissample
buffer for reduction and denaturation." Cell and extracellular ma- cumulation of the antibody caused by overlapping of neightrix extracts were preparedas d e ~ r i b e d . ~The
~ . ~cells
' were washed
bor plasma membranes. Specificity of the anti-uPAR anti2 times withserum-free M199; then they were incubated for 10
body was shown by the lack of staining with nonimmune
minutes at 20°C with PBS containing 0.5%Tx-100, trasylol (800
mouse IgG (Fig 2G), and by the sameuPAR antibody used
KIU/mL), and 2.5%2-mercaptoethanol(2-ME)to remove thecells.
on ECs treated with PI-PLC before immunostaining. As
Wells were then washed once with serum-free M 199, and the reshown in Fig ZH,the majority of the staining was removed
maining material (extracellular matrix) was removed with
60 pL of
from the EC monolayer after PI-PLC treatment.
tris-buffered saline (TBS) containing 1% sodium dodecyl sulphate
Light microscopy analysis of plg-uPA-treated ECs. To
(SDS), trasylol (800 KIU/mL) and 2.5% 2-ME. Sample were then
mixed with denaturing and reducing electrophoresis sample buffer.study the effect of plm formation on theintegrity of the EC
In some experiments, cACA was present during the incubation and monolayer, we treated these cells with plg and uPA. This
type of study is made difficult by the high concentration of
was added together with plg. Radioactivity was measured in a
y
counter, samples fractionated in SDS-polyacrylamide gel electroPAL 1 produced by ECsin vitro. To obtain a detectable cellphoresis (PAGE)
(10%acrylamide)and dried gels autoradiographed surface activity, concentrations over 40 nmol/L uPA and
at -70°C. The autoradiogramswere scanned and quantitated using over 0.3 pmol/L plg are required.16 In preliminary experia computerized image analysis system (RAS 3000; Loats System,
ments in which a plg dose-response curve was tested (range
Wemnister, MD).
0.54 to 1 pmol/L), we have determined that a morphologic
RESULTS
Immunofluorescence analysis of EC monolayers. We
first investigated the presence and localization of theuPAR
by immunofluorescence analysis using specific anti-uPAR
MoAb."
Confluent ECs seeded on gelatin-coated coverslips were
stained with anti-uPAR and antivinculin antibodies. The
primary anti-uPAR antibody and the rhodamine-labeled
rabbit-antimouse IgG were given before cell permeabilization. Only after thisstep, cells weredetergent permeabilized,
incubated with antivinculin antibody followed by fluorescein-labeled goat-antimouse IgG. uPAR was found in distinct patches reminiscent of the focal contacts (Fig 1, A and
C). Thisis confirmed by the uPAR codistribution with vinculin (Fig 1, B and D), similar to what was previously observed in a human fibrosarcoma cell line and in human fibroblast~.'~*~~*~'
lack The
of colocalization of some vinculin
strands (Fig 1, small arrows), indicates specificity ofthe double staining. Codistribution with vn-receptor also shows the
presence of uPAR in focal contacts (Fig 1, E and F). Here
the doublelabeling was performed using anti-uPAR MoAb
and anti83 rabbit serum. When the anti-fi3 rabbit serum
was substituted by nonimmune rabbit serum in double
label
with uPAR, no focal contact strands were present (Fig 1,
G and H). Confluent cells were also analyzed by doubleimmunofluorescence labeling for uPAR and PECAM.
PECAM can be considered a specific marker of endothelial
cell-cell contact sites.48 In thiskind of experiment,the
primary anti-uPAR MoAb was added to living ECs. After
fixation, cells were incubated with fluorescein-labeled goatantimouse IgGfollowed by the addition of rabbitantiPECAM IgG and then rhodamine-labeled swine-antirabbit
IgG; finally, cells were detergent permeabilized (see Materials and Methods). uPAR was found both at focal (small arrows) and cell-cell contact sites (Fig 2 , A and C, large arrows)
effect of plg-uPA treatment was visible at 0.8 rmol/L plg
and 50 nmol/L uPA (not shown). Addition of plg and uPA
to confluent EC generated a loss of the monolayer integrity
as shown by light microscopy analysis of 90-minute-treated
cells, whereas the untreated EC monolayer incubated in serum-free medium was unchanged (Fig 3, compare A with
B). This effect increases very slowly and after 6 hours the
monolayer forms lacunar-like structures (not shown) that
appear because of retraction of the cells. The effect is fully
reversible by substitution of plg-uPA-containing medium
with fresh medium containing serum after 90 minutes (Fig
6 hours (not shown) of treatment. In addi3C), such as after
tion, at 90 minutes, counting of the nuclei at the light microscope did not show any difference between treated and
untreated cells. However, after 6 hours of plg-uPA treatment, the nuclei number decreased by 20% to 40%. This is
probably caused by detachment of cells occurring at late
times. The detached cells were 50% dead, by trypan blue
staining.
In some experiments, ECs were plated on gelatin, fn, vn,
and laminin and grown to confluence over a period of 7 days
in serum-containing medium. After plg-uPA treatment of
the monolayer, cell retraction occurred independently of
the coating protein they had been seeded on (data not
shown), suggesting that this phenomenon was not influenced by the coating protein on which the ECs were plated
and grown for a week in serum-containing medium.
In Table 1 are shown the data comparing the activity of
different PAS in inducing EC retraction. When EC monolayers were treated with pro-uPA instead of uPA, the prouPA was 50-fold more active than uPA on thegeneration of
EC retraction. This finding suggests that a large fraction of
the added uPA was neutralyzed by the PAL1 located beneath the EC monolayer on the cell matrix, as previously
reported by Barnathan et
t-PA (50 nmol/L) was also able to induce EC retraction.
PLASMINOGEN ACTIVATION ON EC SURFACE
997
Fig 1. Double-immunofluorescencelabeling of uPAR and vinculin or vnreceptor. Cells were fixed and incubated (A and C) with R4. anti37'C. followed by rhodamine-conjugatedrabbit-antimouse lgG, then they were penneabilized(see
uPAR MoAb ( 5 pg/mL) for 3 0 minutes at
Materials and Methods). For double labeling, these same cells were incubated (B and D) with MoAb antivinculin for 3 0 minutes at 37'C.
followed by fluorescein-conjugated goat-antimouse IgG. Two representative fields are shown from specimens of two independent experiments. Other EC samples were incubated with anti-O3 (F) or nonimmune rabbit sera (H) (diluted 1 :l 00) for 30 minutes at 37'C. then, for
double labeling, they were bothincubated (E and G) with R4, anti-uPAR MoAb ( 5 pg/mL) for 20 minutes at 4'C before cell fixation. Cells
were then washed, fixed, and incubated, first with fluorescein-conjugated goat-antimouse IgG, to detect the uPAR, then with rhodamineconjugated swine-antirabbit IgG to detect the B3 subunit of the vnreceptor. Finally they were permeabilized(see Materials and Methods).
Small arrows indicate where uPAR and vinculin in focal contacts are shifted, large arrows where they codistributed. Bar, 20 pm.
998
CONFORTI ET AL
Fig 2 . Double-immunofluorescencelabeling of uPAR and PECAM. ECs plated on gelatin-coated coverslips were grown to confluency.
The cells were incubated (A, C, E, and H) with R4, anti-uPAR MoAb ( 5 pg/mL). The cells shown in H were treated with PI-PLC (0.5 U/mL).
before the addition of the uPAR antibody. Control cells were incubated (F) with MoAb anti-ICAM-l (diluted 1: 5 ) and (G) with nonimmune
mouse lgG (5 pg/mL). All primary antibodies were added to the cells at 37'C for 30 minutes before cell fixation. Cells were fixed then
incubated with fluorescein-conjugated goat-antimouse IgG (A,C, E, and G) and with rhodamine-conjugatedrabbit antimouse IgG (H and F).
For double labeling, some
of the samples previously labeled for uPAR were incubated with anti-PECAM-rabbit IgG (2.5 rg/mL) (B). nonimmune rabbit IgG (2.5 pg/mL) (D) for 30 minutes at 37°C. followed by rhodamine-conjugated swine-antirabbit IgG. Finally, all the samples
were detergent permeabilized(see Materials and Methods). The ECs shown in (E) were used at subconfluency. Small arrows indicate focal
contacts, large arrows indicate cell borders. Bar, 2 0 pm.
PLASMINOGENACTIVATIONON
999
EC SURFACE
Treated
Untreated
EC monolayers grown in 24-well plates were mainFig 3. plg-uPA treatment induces changes of EC-monolayer morphology. Confluent
tained in serum-free M199 for 50 minutes, then treatedwith plg (0.8pmol/L) anduPA (50 nmol/L) as described in Materials and Methods.
(A) plg-uPA treated
cells; (B) the corresponding untreated control maintainedin serum-free medium. Photographs of the monolayers were
taken after 90 minutes from the addition ofuPA, on living cells. Two representative fields of plg-uPA-treated cells are shown. (C) The
1 hour after removal of plg-uPA medium and
recovery ofEC-monolayer integrity, after 90 minutes of plg-uPA. The photograph was taken
cells.80 pm.
substitution with fresh M199 containing serum. Arrows indicate sites of discontinuity between Bar,
However, considering the specific biologic activity of the
two proteins used, tPA (792,000 to 704,225 IU/mL) versus
uPA (100,000 IU/mL), we have calculated that uPA was at
least IO-fold more efficient in inducing EC retraction. Interestingly, when we used the low molecular-weight uPA (33
Table 1. Comparison of Different PAS
in Inducing €C Retraction
PAS
nmol/L
IU/Well
uPA
15
pro-uPA
tPA
Low molecular weight uPA
50
1
50
100
144-161"
32
-
Confluent EC monolayersin 96-wellculture dishes were proteolitically
treated as described in Materials and Methods. Retraction was studied
by the addition of plg in serum-free medium, followed after 10 minutes
by the addition of the PAS(reactionvolume = 6 0 pL). The concentration
range tested for each PA was 0.1 t o 100 nmol/L. The table reports the
minimal dosefor each PA ablet o induce cell retraction at the same time
and at a degree comparablewith 5 0 nmol/L uPA, consideredthe positive
control. The comparison began when cellstreated with 50 nmol/L uPA
started to retract. Cells were observed every 15 minutes for 1 hour. At
the last time, they were fixed and stained (see Materials and Methods).
Samples were run in duplicate, in two independent experiments with
comparable results.
Two batches of tPA were used for each experiment.
kD), in which the uPAR binding site is missing, a twofold
amount was required to obtain the EC retraction effect observed with the 50 kD uPA.
Plasmin itself (0.8 pmol/L) could induce EC retraction
when added to EC monolayer.
The cell retraction we have found with ECs was also observed when human saphenous ECs were used, suggesting
that this is not a specific property of ECs,but that it is common to the endothelium from different vascular districts.
Duplicate samples were run in two independent experiments giving the same results (data notshown).
Quantitation of EC-monolayer retraction by measure of
monolayer permeability. Toquantitate the loss ofEC
monolayer integrity, we measured the permeability to '251HSA using a Transwell permeability system. Confluent ECs,
grown on fibronectin-coated filters in the upper compartment of the transwell chamber, were plg-uPAtreated in serum-free medium supplemented with I2'I-HSA (see Materials and Methods). At different times, samples were
withdrawn from the lower compartment and theradioactivity, passed from the upper to the lower compartment,
counted. plg-uPA treatment increased EC-monolayer permeability starting within 20 to 70 minutes from the addition
of uPA to the monolayer, whereas the single components
had no effect (Fig 4A). The effect was dependent on uPA
concentration (Fig 4B). For each point of the kinetic and
CONFORTI ET AL
"1 A
plg-uPA treatment itself did not modify the fn-coated filter permeability. In fact, in the absence of cells the permeability of I2%HSA wassimilar for the untreated or plg-uPAtreated fn-coated filters. Nor was 1251-HSAdigested during
the time of incubation, asassessed bySDS-PAGE and autoradiographic analysis of I2'I-HSA samples collected at
different times (data not shown).
Cell-surfaceboundplm is involved in the loss ofEC-monolayer integrity by plg-uPA. Several data have shown that
surface-bound plrnis resistant to a2-anti-plm, as lysinebinding sites are required for both cell-surface and az-antiplm
Trasylol, on
the
other
hand, does not discriminate between soluble and lysine binding site-bound
0
60
80 100 120 140 160
plm. Figure 5 shows a typical experiment in which the increased EC permeability caused by the plg-uPA treatment
Time (min)
could be 60% inhibited by a2-anti-plm at 150 minutes but
was fully inhibited by trasylol. At 90 and 120 minutes, the
plg-uPA-treated samples compared in the presence and ab15
sence of trasylol gave P values of .05 and .03, respectively.
The inset of the figure shows ECs grown on filters, fixed, and
stained at the end of the experiment (after 150 minutes of
plg-uPA treatment). cY2-anti-plm could only partially pro10
tect the monolayer by plg-uPA treatment compared with
trasylol. On the other hand, both a2-anti-plm and trasylolinhibited
plm in solution over 90% as measured on super5
natant samples taken at the end ofthe
experiment and tested
by a chromogenic substrateassay (S225 1) (data not shown).
In three independent experiments, the
average inhibition by
0
k
7
SE
at
150
minutes
with a P value of
cyanti-plm
was
64
0
20
40
60
80
100
.07. Thus, surface-bound plrn may be involved in theloss of
[uPA] nM
EC-monolayer integrity.
A role for lysine binding sites in the loss of cell-cell conFig 4. Quantitative analysis of the time and uPA concentrationtacts
was also supported by additional light-microscopy data
(A) TimedependentgenerationofEC-monolayerpermeability.
in which plg-uPA treatment was performed in the presence
course. (Cl) plg-uPA treated cells; (m) treated cells with uPA alone;
or (0)with plg alone. (B) uPA dose-response. The cellswere mainof 5 mmol/L tACA, which competes with the cell surface
tained in serum-free medium for
50 minutes beforethe addition of
for plg binding. As shown in Fig 6, incubation of cells with
the same amountof plg (0.54rmol/L) followed after 10 minutes by
plg in the presence of 5 mmol/L cACA followed by the adincreasing amountsof uPA. At the indicated time (A) and after 120
dition of uPA, strongly decreased the effect of the plg-uPA
minutes (B) from the addition of uPA, l00 #L of samples (of 600
treatment (compare C with B). In this experiment, a conpL) were withdrawn from the lower compartment of the transwell
chamber and counted in ay counter (see Materials and Methods).
centration of 100 nmol/L uPA was used to obtain a strong
exEach point(A and B) is the mean of duplicates from one typical
effect and to better visualize the inhibition.Combining
periment. The background radioactivity of untreated cells, maintACA and a2-anti-plm resulted in a further reduction of the
tained in serum-free medium,
has been subtracted from each curve.
plg-uPA
effect, although the inhibition was still not comthan 10%and
Duplicates of plg-uPA-treated samples diRer by less
plete (Fig 6, D). In the Transwell system under the same
of untreatedsamples by less than 5%.
experimental conditions, tACA inhibited by 53% the plguPA-induced permeability (not shown).Data of'"I-plg
dose-response curves, the standard error calculated on the
cleavage by uPA in the presence of tACA also strongly indimean of two independent experimentsrun in duplicatewas
cate that cell surface plays an important role in the modifiless than 10%.
cation of the EC monolayer caused by plg activation (see
In some experiments, the passage ofI2%HSA through the
below).
fn-coated filters, in the presence or absence of cells, was
Functional role of receptor-bound uPA in the loss of ECmeasured. Fn-coated filters were incubated with or without
monolayer integrity. Activation of plg byendogenous uPA
cells in 20% NCS-containing M 199 medium for 1 week, unis totally dependent on the cell surface.13 To directly test
til cells reached the confluence. Then cells or filters were plgwhether exogenously added receptor-bound uPAwas inuPA-treated and the passage of "'I-HSA measured. In the
volved in EC retraction, the enzymatically inactive ATF of
absence of cells, the passage of "'I-HSA through the filters
uPA, which competes with uPA for bindingto receptor, was
was faster; within the first 10 minutes, we measured IO-fold
used as an antagonist of the plg-uPA effect on EC. In the
more '251-HSAin the lower compartment of the transwell
monolayer permeability assay, ATF inhibited plg-uPA-inchamber compared to filters with cells. At 120 minutes,
duced HSA permeability by 66% at 90minutes as shown in
when the EC monolayer was retracted, a fivefold difference
a typical experiment (Fig 7). The average inhibition by ATF
was observed.
"1
B
1001
PLASMINOGEN ACTIVATIONON EC SURFACE
H
X
E
+ a2-antiplasmin
0
+ trasylol
8-
Q
S
a
U)
6-
I
v)
cu
7
4-
LC
0
2-
0
0
20
40
60
80
100
Time (min)
120
140
160
Fig 5. Effect of protease inhibitors on EC-monolayerpermeability. Confluent EC monolayers grown on fibronectin-coated transwell filters
were incubated with 0.54 pmol/L plg in presence of lZ51-HSA(1Og cpm/well). After 10 minutes 50 nmol/L uPA was added. ( 0 )No inhibitors;
(m) n,-anti-plm; (0)trasylol. Both inhibitors were used at a molar concentration (1.62 pmol/L) threefold excess over plg. EC-monolayer
Permeability was measured at different times counting the lz5l-HSAcontained in 100 pL of samples (out of 600 pL) withdrawn from the
lower compartment of the transwell chamber. Each point represents the mean of duplicates. The background radioactivity of untreated
cells, maintained in serum-free medium, has been subtracted from each curve. Duplicate of plg-uPA-treated samples in the presence or
absence ofaianti-plrn differ by lessthan 10%. Untreated samples or plg-uPA-treated samples in thepresence of trasylol differ by lessthan
5%. Inset shows EC monolayers at theend of the experiment (1 50 minutes of plg-uPA treatment). Photographs were taken on cells fixed
with diff-quik and stained with crystal-violet. Bar, 100 pm.
in four independent experiments was 46% k 9% SE at 90
minutes with a P value of .07. ATF added to theuntreated
EC did not modify EC monolayerpermeability (not shown).
Light microscopic assessment of EC morphology was performed in parallel dishes. Figure 7, inset, shows that after 90
minutes of EC treatment with plg-uPA, ATF could almost
entirely prevent morphologic changes (A v C). However, the
inhibitory effect of ATF was transient because visible ECmonolayer damage became apparent at 150 minutes (B v
D). The addition of cu2-anti-plm,which inhibits plmactivity
in solution together with ATF, which inhibits cell-surface
plm generation, raised the percentage of inhibition up to
75% at l50 minutes (not shown). These data show that cellsurface binding of uPA is an important prerequisite in plguPA-induced EC retraction.
Plg is uctivuted on EC surface. To test whether plg activation took place on thecell surface under our experimental
conditions, we incubated ECs with a plg concentration able
to generate EC-monolayer retraction, 0.8 pmol/L, but also
containing 1.5 X lo6cpm 1251-plg.After 10 minutes, the radiolabeled mixture was removed and cell-bound IZ5I-plg
chased by the addition of 0.8 pmol/L unlabeled plg (corresponding to 200-fold molar excess ofthe cell-bound I2%plg,
see Materials and Methods), .then incubated for further 15
minutes at 37"C, in the presence of 50 nmol/L uPA. Supernatants were then removed, and cell and matrix extracts
prepared as described under Materials and Methods. Samples were analyzed by SDS-PAGE under reducing conditions and autoradiography. As shown in Fig 8A, plm was
generated in the presence of uPA with bands at about 72
CONFORTI ET AL
1002
untreated
+ EACA
plg-uPA treated
+ E ACN% -antiplasmin
Fig 6. Role of lysinebinding
sites in cell-associated proteolysis.€C were grown at confluency on 0.5% gelatin-coated microtiter wells and treated with
plg-uPAin serum-free medium
as describedin the legend to Fig
3. (A) Untreated €C moodayer;
(B) plg-uPA-treated cells. In (C)
and (D),cells were incubated, respectively, with 5 mmol/L tACA
or 5 mmol/L rACA plus 2.4
pmol/L a,-anti-plm, which were
added alongwith 0.8 pmol/L plg,
10 minutesbeforeadditionof
uPA (100 nmol/L). Photographs
were taken after 2 hours of treatment on fixed and stained cells
(see
legend
to Fig 5). Bar,
100 Mm.
and 27 kD in the supernatant (lane l), in the cell-associated
agreement with previous data." Interestingly, in our experfraction (lane 2), and in the matrix-associated fraction(lane
imental system, thesedata show that in presence of cACA,
3). By far, the highest efficiency of plrnformation was in the
when most of the plrn is present in the phase solution, EC
cell-associated fraction. Interestingly, when unbound "'lretraction was strongly inhibited (Fig 6C),
suggesting again
the importance of cell-associated plrn in the generation of
labeled plg was not chased with the unlabeled competitor
plg (Fig 88). the cell-surface preference for plrn formation
this phenomenon.
disappeared (lanes 4 to 6). By 'zsI-plgcleavage assay,we calDISCUSSION
culated that 92%of cell-associated plg was activatedby 50
nmol/L uPA within I5 minutes. This value was determined
In this report, we have shown that uPAR in EC is localby densitometric scanningof the autoradiogram measuring ized at cell substratum aswell as at cell-cell contacts (Figs1
the intensity of the bands correspondingto the high and low
and 2). In focal contacts, uPAR colocalized with uPA (data
molecular-weight plrn chains or to the uncleaved plg, after
not shown), vinculin, and vn receptor. The colocalization
samples fractionation in SDS-PAGE and autoradiography
of uPAR with vn receptor has alsobeen found for human
(Fig SB, lane 5 ) . As also shown previously by other investirhabdomyosarcoma cells, embryonic skin fibroblast, and
gators, bound Izsl-plg partially dissociated from cell bindingadherent U937 cell^.^' uPAR codistributed with PECAM at
sites49; however,the presence of excess unlabeled competicell-cell contacts, suggesting that the two molecules are astor plg (Fig SA) rules out the possibility that 12SI-plmwas
sociated to similar junctional structures. Wehave also
formed in solution from the released 12sI-plgand subseshown that treatment of EC with plg-uPA generates morquently rebound to the cells. Therefore, we conclude that
phologic and functional changes of EC monolayer (Fig 3)
under our experimental conditions, plrn formation takes
with a retraction accompaniedby an increasein monolayer
place onthe cell membrane.
permeability to high molecular-weightsolubleproteins
IzsI-plg cleavage by uPAin presence of 5 mmol/L cACA
(HSA).
The EC-retraction phenomenon was not influenced by
inhibited the interaction ofplg with the EC by 85%.Furthermore, in the presence of tACAthe plg was 100%converted
the coating protein on which the cells were plated (fn, vn,
to plrn in cell, matrix, and fluid-phase-associated fractions
laminin, or gelatin). This is not surprisingbecause, in such
(not shown), whereasin the absence of cACA some plg reexperiments, although the cellswereplatedondifferent
mained partially uncleaved. under the same experimental
coating proteins, they were then cultured for several days
conditions(Fig S, lanes 4 through 6). These results highlight in serum-containing medium before
the plg-uPA treatment.
two important points: first, that the inhibition mechanism
We believe that during the time ofcell culture, they produce
of EC retraction by tACA may be at least in part, related to
and secrete their own extracellular matrix proteins; proteins
from serum can also
be adsorbedon the initial coating,both
the inhibition of the plg-binding to the cells: second, that
tACA-boundplgismoreefficientlyactivated
to plm, in
contributing in making the final matrix quite similar. Our
PLASMINOGENACTIVATIONON
EC SURFACE
1003
90 min
Fig 7 . Effect of ATF on ECmonolayer permeability. EC
were
grown
to confluency
on fibronectin-coated transwell
filters (see Materials and Methods). Cells were plg-uPA
treated in serum-free medium
as described in legend to Fig 3.
ATF (1.2 pmol/L) was added together with plg (0.54 pmol/L),
10 minutes before the addition
of uPA (50 nmol/L). (Bottom) (0)
plg-uPA-treatedcells;
(0) plguPA-treated cells in presence of
ATF. At different times, 100 pL
of samples were counted in a y
wunter (seelegend to Fig 5).
Each point represents the mean
of duplicates which differ by less
than 10%.Thebackgroundradioactivity of untreated cells,
maintained in serum-free medium, has been subtracted from
of uneachcurve.Duplicate
treated cells differ by less than
5%. (Top) Morphologic analysis
of cells performed
in parallel, and
pictures
taken
on
fixed and
stainedcells
at the indicated
time. Bar, 100 pm.
data, in which laminin and fn were found on cellscultured
on gelatin,support this hypothesis (data not shown).
The retraction effectiscausedbyplrn
formation, as
shown by the inhibition by trasylol and a2-anti-plm. However,whereastrasylolcouldtotallyinhibit
the plg-uPA
effect, n2-anti-plmwas only partially inhibitory, indicating
that a quota of plrn is generated at the cell surface. In fact,
surface-bound plrn is protected from the a2-anti-plm action, but not fromtra~ylol.~~."
Moreover, tACA.a molecule
that prevents plg binding to the cell surface by competing
with cells forplg lysine-binding sites" could also
inhibit the
effect of plg-uPAtreatment. Preferential activation of plgon
the cell surface was also shown by direct experiments (Fig
8).
The plrn formation oncell surface depends on the binding
of uPA to uPAR becausethe uPAR antagonist, ATF, inhibited both functional and morphologic effects. The effect of
plg-uPA on EC is not accompanied by an indiscriminate
150 min
40
60
Time (min)
80
100
proteolysis of cell-cell and focal-contact molecules.In fact,
immunofluorescence experiments failed to show any major
changes in the distribution of uPAR, vn receptor, and
PECAM (data not shown). The fact that tPA could alsoinduce EC retraction was not surprising, because t-PA has
beenshownpreviously
to activate cell-bound plg more
efficiently than fluid-phase pig." Moreover, receptors fortPA have been identified on ECS.'~However, the plg-activation efficiency of the tPA was IO-fold lower than the uPA,
and it was much lower if compared with the pro-uPA. A
possible explanation for this canbe the intrinsic characteristic ofthe tPA that requires fibrin as cofactor
to efficiently
induce plg activation.
Because receptor for plg can also bind plm," the EC retraction induced by the addition of plrn itself strongly indicates that this isa plm-mediated event.In the case ofthe low
molecular-weight uPA, plrn formation in fluid phase could
also be the cause of EC retraction. We believe that some of
1004
-
CONFORTI ET AL
A
The morphologicandfunctionaleffectsare
wellexplained by the localization ofuPA and uPAR, as EC retraction and monolayerpermeabilitymay be logicalconsequences of proteolyticeventsoccurring at cell-cell and
cell-substratum contacts.
uPA and uPAR have been shown to be essential in the
dissemination of tumor cells in a variety of modelsystem~.~"'The findings reported in this report open the way
to a molecular studyof the role of the cell-surface plg activation in extravasation.
ACKNOWLEDGMENT
We thank Dr J.Henkin for providing ATF.
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1 2 3
4 5 6 7
8
Fig 8. Effect of ECson plasminogencleavage by uPA. Confluent
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