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Matrix metalloproteinase 12dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 10, October 2004, pp 3275–3285
DOI 10.1002/art.20562
© 2004, American College of Rheumatology
Matrix Metalloproteinase 12–Dependent Cleavage of Urokinase
Receptor in Systemic Sclerosis Microvascular Endothelial Cells
Results in Impaired Angiogenesis
Silvia D’Alessio,1 Gabriella Fibbi,1 Marina Cinelli,1 Serena Guiducci,1 Angela Del Rosso,1
Francesca Margheri,1 Simona Serratı̀,1 Marco Pucci,1 Bashar Kahaleh,2 Pansheng Fan,2
Francesco Annunziato,1 Lorenzo Cosmi,1 Francesco Liotta,1
Marco Matucci-Cerinic,1 and Mario Del Rosso1
shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is
known to impair uPAR functions. These properties of
SSc MVECs were associated with poor spontaneous and
uPA-dependent invasion, proliferation, and capillary
morphogenesis. The uPAR cleavage occurring in SSc
MVECs was associated with overexpression of MMP-12.
SSc MVEC–conditioned medium impaired uPAdependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a
general hydroxamate inhibitor of MMP activity and
anti–MMP-12 antibodies restored this SSc MVEC–
induced impaired functioning.
Conclusion. Overproduction of MMP-12 by SSc
MVECs accounts for the cleavage of uPAR and the
impairment of angiogenesis in vitro and may contribute
to reduced angiogenesis in SSc patients.
Objective. Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse
form of systemic sclerosis (SSc). The present study was
undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs)
in the expression of the cell-associated urokinase-type
plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process.
Methods. MVECs were isolated from the dermis
of healthy individuals and from the dermis of patients
with diffuse SSc. The uPA/uPAR system was examined
at the protein and messenger RNA levels. Angiogenesis
was assayed on Matrigel-coated porous filters and
plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity
of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting.
Results. Compared with MVECs from healthy
skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as
Systemic sclerosis (SSc) affects the skin and the
internal organs, resulting in tissue fibrosis and organ
dysfunction. The pathogenesis of SSc is characterized by
3 distinct processes: 1) microvascular alterations, including capillary endothelial cell injury, 2) perivascular
extravasation of lymphocytes, and 3) accumulation of
collagen in the dermis. The microvascular alterations
usually precede the appearance of clinically detectable
fibrosis. Capillaroscopy has shown that microvascular
modifications are characterized by megacapillaries and
eventual desertification of the nailfold (1).
The mechanism of microvascular injury is still
unknown. Some authors report that sera from patients
with acrosclerosis potentiate the angiogenic capability of
normal human mononuclear cells (2,3). In diffuse SSc, a
subset of mononuclear cells displays enhanced angio-
Supported by grants from the Scleroderma Foundation (004/
01) and the Ministero dell’Istruzione, Università e Ricerca (Progetti di
Ricerca di Interesse Nazionale).
1
Silvia D’Alessio, PhD, Gabriella Fibbi, PhD, Marina Cinelli,
PhD, Serena Guiducci, MD, Angela Del Rosso, MD, Francesca
Margheri, PhD, Simona Serratı̀, PhD, Marco Pucci, PhD, Francesco
Annunziato, PhD, Lorenzo Cosmi, MD, Francesco Liotta, MD, Marco
Matucci-Cerinic, MD, Mario Del Rosso, MD: University of Florence,
Florence, Italy; 2Bashar Kahaleh, MD, Pansheng Fan, PhD: Medical
College of Ohio, Toledo.
Address correspondence and reprint requests to Mario Del
Rosso, MD, Professor of General Pathology, Department of Experimental Pathology and Oncology, Viale G. B. Morgagni, 50, Florence
50134, Italy. E-mail: delrosso@unifi.it.
Submitted for publication February 3, 2004; accepted in
revised form June 30, 2004.
3275
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D’ALESSIO ET AL
genic activity (4). In contrast, impaired angiogenic activity has been reported in peripheral blood lymphocytes
and monocytes in the diffuse subset of SSc (5,6), although Ribatti et al (7) have shown that angiogenesis is
stimulated on the chick embryo chorioallantoic membrane by skin biopsy tissue from patients with diffuse
SSc. Circulating endostatin concentrations are significantly increased in patients with SSc, and this may
contribute to an overall decrease of angiogenesis in the
course of the disease (8).
Purportedly, microvascular endothelial cells
(MVECs) can perform neoangiogenesis only when provided with the proper enzymatic machinery, enabling
them to lyse extracellular matrix (ECM) and to enter the
surrounding tissue. The cell-associated plasminogen activator system is known to play a crucial role in the
angiogenesis process by modulating the adhesive properties of endothelial cells in their interactions with ECM
and in the degradation of ECM (9–13). We have demonstrated that urokinase-type plasminogen activator
(uPA) induces neovascular growth in the avascular
rabbit cornea and promotes growth, chemotaxis, and
matrix invasion of endothelial cells in vitro, by interaction with its receptor, uPAR (11). The uPA required for
invasion can be produced by endothelial cells themselves, as well as by other cells of the microenvironment
in which angiogenesis develops, such as fibroblasts and
macrophages.
In this study, we isolated MVECs from the
dermis of normal, healthy individuals and from the
dermis of patients with diffuse SSc, to study the relationship between angiogenesis and the endothelial cell uPA/
uPAR system. Our results indicate that SSc MVECs are
prevented from entering an angiogenic program by
matrix metalloproteinase 12 (MMP-12)–dependent
cleavage of domain 1 (D1) of uPAR, a structural modification that prevents the functioning of uPAR in
endothelial cell invasion and proliferation.
PATIENTS AND METHODS
Antibodies and reagents. Anti-CD87 mouse monoclonal antibody (mAb), which identifies total uPAR, mouse mAb
anti–uPAR domain 1, and mouse mAb anti–uPAR domain 2
were purchased from American Diagnostica (Montreal, Quebec, Canada). Mouse mAb 5B4, which recognizes the kringle
domain of the A-chain of uPA and prevents uPA–uPAR
interaction (11,14), was a kind gift from Dr. M. L. Nolli
(Lepetit Research Center, Varese, Italy). Rabbit mAb against
the hinge region of MMP-12 was from Chemicon International
(Temecula, CA). The uPA was obtained from Serono (Rome,
Italy), and endothelial cell growth supplement (ECGS) was
purchased from Calbiochem (Nottingham, UK). Reagents for
messenger RNA (mRNA) studies were from Promega Italia
(Milan, Italy), and other reagents for cell culture, including
fetal calf serum (FCS), were purchased from Sigma (St. Louis,
MO). Diff-Quick was from Mertz-Dade (Dade International,
Milan, Italy). Matrigel was obtained from Becton Dickinson
(Bedford, MA).
Cells. Patients with SSc and healthy controls were used
as the sources of MVECs. MVECs were isolated from biopsy
samples of involved skin of the hands of 6 SSc patients who
underwent skin biopsies for diagnostic purposes at the Department of Medicine (Section of Rheumatology) of Florence
University, and from 15 healthy patients undergoing surgery
for trauma to the hands. The study was approved by the local
ethics committee, and written informed consent was obtained
from each subject enrolled. The patients were not receiving
steroids, cyclophosphamide, D-penicillamine, relaxin, or other
disease-modifying drugs. Calcium channel blockers were
stopped 10 days before the biopsy. Only proton-pump inhibitors and cisapride were allowed.
To obtain the cells, skin biopsy samples were mechanically cleaned of epidermis and adipose tissue in order to
obtain a pure specimen of vascularized dermis, and were
treated as described elsewhere (15). In some cases, clusters of
round-shaped cells were squeezed from microvessels and
formed colonies composed of polygonal elements. Such colonies were detached with EDTA, and CD31-positive cells were
subjected to immunomagnetic isolation with Dynabeads CD31
(Dynal ASA, Oslo, Norway) (16). Isolated cells were further
identified as MVECs by labeling with anti–factor VIII–related
antigen and by reprobing with anti-CD31 antibodies. Cells
were maintained in complete MCDB medium supplemented
with 30% FCS, 20 ␮g/ml ECGS, 10 ␮g/ml hydrocortisone, 15
UI/ml heparin, and antibiotics (100 UI/ml penicillin, 100 ␮g/ml
streptomycin, 50 ␮g/ml amphotericin). MVECs from healthy
individuals and from SSc patients were used between the third
and tenth passages in culture.
Enzyme-linked immunosorbent assays (ELISAs) for
uPA, uPAR, and uPAR fragments. The IMUBIND total uPAR
ELISA kit (American Diagnostica) was used to determine the
expression of uPAR in aliquots of cell lysates. The D1 domain
of uPAR was measured by a modification of the total uPAR
ELISA kit, using murine mAb anti-human uPAR D1 as the
capture antibody. Quantification of uPAR D1 was carried out
by comparing the values with those of a standard curve, while
uPA was quantified by U-PA Actibind ELISA (Technoclone,
Wien, Austria).
Flow cytometry analysis. Flow cytometry analysis on
MVEC suspensions was performed as described previously
(17,18). Briefly, 3 ⫻ 105 cells were suspended in phosphate
buffered saline (PBS) containing 0.5% bovine serum albumin
and 0.02% sodium azide, and after saturation of nonspecific
binding sites with total rabbit IgG, cells were incubated for 20
minutes with specific or isotype control mAb. Cells were then
washed and incubated with a fluorochrome-conjugated antiisotype antibody for an additional 20 minutes. Finally, cells
were washed and analyzed on a BD-LSR cytofluorimeter
(Becton Dickinson).
Migration assays. The Boyden chamber was used to
evaluate spontaneous and stimulated invasion (chemoinvasion) of cells through Matrigel-coated porous filters, as described previously (11,14). For assessment of spontaneous
IMPAIRED SSc MVEC ANGIOGENESIS BY MMP-12 CLEAVAGE OF uPAR
invasion, 50 ␮l of cell suspension (6.25 ⫻ 103 cells) was placed
in the upper compartment of the chamber, and migration was
allowed to occur for 6 hours. For assessment of chemoinvasion,
test solutions containing increasing concentrations of uPA
(5–500 ng/ml) were dissolved in serum-free medium and
placed in the lower wells. In the experiments with neutralizing
antibodies, the anti-uPA mAb (1.5 ␮g/ml) was added to the
lower well with the test substance (uPA), while the anti-uPAR
mAb (1.5 ␮g/ml) was incubated with the cell suspension.
Irrelevant mouse IgG was used to verify the specificity of the
effect. The number of cells moving across the filter was used as
the measure of mobilization. Experiments were performed in
triplicate. Migration values were expressed as the mean ⫾ SD
number of total cells counted per filter, or as a percentage of
the basal response value.
Proliferation studies. Cell growth was quantified in
subconfluent cell monolayers, as described previously (11,14).
MVECs were seeded onto 24 multiwell plates (Sarstedt,
Verona, Italy) (15 ⫻ 103 cells/well) in complete MCDB
medium, and were left to adhere overnight. Cells were then
extensively washed in PBS and maintained for 24 hours in 1%
FCS–MCDB medium. Medium was removed and cells were
incubated with 1% FCS–MCDB medium containing increasing
concentrations of uPA for 48 hours. The proliferative effect of
30% FCS was defined as the optimal growth. In the experiments with neutralizing mAb, cells were challenged with uPA
in the presence of the mAb (1.5 ␮g/ml). Irrelevant mouse IgG
was used as a negative control. Each experimental point was
performed in triplicate. At the end of the incubation, cells were
fixed by adding 1 ml of ice-cold methanol, stained with
Diff-Quick, and counted. Values were expressed as the percentage increase or decrease over the basal response value.
Detection of mRNA transcripts by semiquantitative
reverse transcription–polymerase chain reaction (RT-PCR),
and quantification of PCR products. Levels of mRNA for
uPAR and uPA genes in normal, healthy MVECs and SSc
MVECs were determined by an internal-based semiquantitative RT-PCR, using procedures and primers previously described (19). For MMP-12, 2 ␮l of reverse-transcribed DNA
was amplified, using MMP-12–specific sense (5⬘-CCA-CTGCTT-CTG-GAG-CTC-TT-3⬘) and antisense (5⬘-GCG-TAGTCA-ACA-TCC-TCA-CG-3⬘) primers, or GAPDH-specific
sense (5⬘-CCA-CCC-ATG-GCA-AAT-TCC-ATG-GCA-3⬘)
and antisense (5⬘-TCT-AGA-CGG-CAG-GTC-AGG-TCCACC-3⬘) primers as a control. Semiquantitative PCR was
performed for 35 cycles at 58°C in a thermocycler. The
reaction products were analyzed by electrophoresis in 1%
agarose gel containing ethidium bromide, followed by photography under ultraviolet illumination using Polaroid positive/
negative instant films, and quantified as reported elsewhere (19).
In vitro capillary morphogenesis assay. Matrigel (0.5
ml; 10–12 mg/ml) was pipetted into 13-mm (diameter) tissueculture wells and polymerized for 30 minutes to 1 hour at 37°C
(20,21). MVECs were plated (60 ⫻ 103/ml) in complete
MCDB medium supplemented with 30% FCS and 20 ␮g/ml
ECGS. Capillary morphogenesis was also performed in the
presence of 1.5 ␮g/ml CD87 mAb (anti-uPAR) or 5B4 mAb
(anti-uPA). Positive controls were obtained upon stimulation
with 10 ng/ml vascular endothelial cell growth factor (VEGF).
Irrelevant mouse IgG was used as a negative control. In other
cases, normal MVECs were incubated with conditioned me-
3277
dium containing SSc MVECs, either alone or supplemented
with antibodies to MMP-12 (10 ␮g/ml). Plates were photographed at 6 hours and at 24 hours. Results were quantified at
6 hours by measuring the percentage of the photographic field
occupied by endothelial cells, as determined by image analysis.
Six to nine photographic fields from 3 plates were scanned for
each point.
Preparation of SSc cell–conditioned medium. Confluent cultures of normal MVECs and SSc MVECs were washed
twice with PBS and incubated overnight in the presence of
MCDB medium supplemented with 2% FCS. The culture
supernatant was centrifuged at 1,500 revolutions per minute
for 10 minutes, and either used immediately or stored at
⫺20°C.
Western blotting, treatment with protease inhibitors,
and anti–MMP-12. Normal MVECs were grown to 70%
confluence and were serum-starved overnight in MCDB medium supplemented with 2% FCS. The cells were then incubated with or without SSc MVEC–conditioned medium for 24
hours, in the presence or in the absence of 10 ␮g/ml of
anti–MMP-12 mAb or protease inhibitors (a general hydroxamate inhibitor of MMP activity, Ilomastat, or GM6001, at 25
␮M [Chemicon International]; a serine-protease inhibitor,
aprotinin, at 1 ␮g/ml [Sigma]; or a selective low molecular
weight inhibitor of cysteine protease, E-64, at 200 ␮M, kindly
provided by Professor Francesco Paoletti from the University
of Florence). Cells were then suspended in lysis buffer (10 mM
Tris HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100,
15% glycerol, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 ␮g/ml
aprotinin). Sixty micrograms of the cell extract protein was
electrophoresed in sodium dodecyl sulfate–12% polyacrylamide gels under reducing conditions and then blotted to a
polyvinylidene difluoride membrane (Hybond-C Extra; Amersham Biosciences, Piscataway, NJ) for 3 hours at 35V.
The membrane was incubated with 5% skim milk in 20
mM Tris buffer, pH 7.4, for 1 hour at room temperature to
block nonspecific binding and then probed with a mouse mAb
directed against the first domain (D1) as well as the second
domain (D2) of uPAR (American Diagnostica, Stamford, CT)
overnight at 4°C. The same procedure was used to perform
Western blotting of aliquots of MVEC culture medium, to
identify MMP-12 with anti–MMP-12 antibodies. After incubation with horseradish peroxidase–conjugated donkey antimouse IgG (1:5,000) for 1 hour (Amersham Biosciences),
immune complexes were detected with the enhanced chemiluminescence detection system (Amersham Biosciences). The
membranes were exposed to autoradiographic films (Hyperfilm MP; Amersham Biosciences) for 1–30 minutes.
Statistical analysis. Results are expressed as the
mean ⫾ SD. Multiple comparisons were performed by the
Student-Newman-Keuls test, after demonstration of significant
differences among medians by nonparametric variance analysis
using the Kruskal-Wallis test. P values less than 0.05 were
considered significant.
RESULTS
Hypofunctionality of the uPA/uPAR system in
SSc MVECs. MVEC expression. Characterization of the
uPA/uPAR system was performed on confluent mono-
3278
D’ALESSIO ET AL
Figure 1. Spontaneous invasion, urokinase-type plasminogen activator (uPA)–induced chemoinvasion, and proliferation of normal, healthy
microvascular endothelial cells (N-MVEC) and systemic sclerosis endothelial cells (SSc-MVEC). A, To assess spontaneous invasion of
Matrigel-coated filters in the Boyden chamber assay, 6.25 ⫻ 103 cells were placed in the upper well and invasion was allowed to occur for 6 hours.
Each blocking monoclonal antibody (mAb) as well as irrelevant mouse IgG were used at 1.5 ␮g/ml. B, uPA-dependent chemoinvasion of Matrigel
in the Boyden chamber. Results are the total number of cells/filter after 6 hours. C, Effect of blocking mAb and of irrelevant IgG on uPA-dependent
chemoinvasion. Results are the percentage variation in relation to the migration of unstimulated cells (control), considered to be 100%. D, Response
of N-MVEC and SSc-MVEC to uPA challenge. E, uPA-dependent proliferation with blocking mAb and irrelevant mouse IgG. Results are the
percentage variation in relation to the number of cells at the moment of plating (150 ⫻ 103/well), considered to be 100%. Bars show the mean ⫾
SD of 3 experiments performed in triplicate in 3 normal and 3 SSc cell lines. ⴱ ⫽ P ⬍ 0.05 versus control.
layers of cells for which CD31 staining yielded positive
results in more than 90% of the cell population. No
difference in cell density, once at confluence, was observed between normal MVECs and SSc MVECs, which
allowed us to perform experiments in conditions of
similar cell density. ELISA determination of uPA in the
culture medium indicated a level of 31.0 ⫾ 9.1 ng/ml in
both cell lines. In contrast, we found that uPAR was
lightly overexpressed in SSc MVECs (15.3 ⫾ 3.0 ng/106
cells in normal MVECs compared with 23.1 ⫾ 4.3 ng/106
cells in SSc MVECs; P ⬍ 0.05). Semiquantitative RTPCR analysis of the specific mRNA confirmed the data
obtained at the protein level (results not shown).
MVEC invasion. Invasion of the surrounding matrix by endothelial cells is a necessary step in the
angiogenic program. Figure 1A shows that normal
MVECs and SSc MVECs were differentially prone to
spontaneous invasion of Matrigel-coated filters. Invasiveness of normal MVECs (650 ⫾ 57 cells/filter) was
about twice that of SSc MVECs (330 ⫾ 35 cells/filter).
IMPAIRED SSc MVEC ANGIOGENESIS BY MMP-12 CLEAVAGE OF uPAR
3279
Figure 2. Capillary morphogenesis in N-MVEC and SSc-MVEC. N-MVEC and SSc-MVEC were plated on Matrigel (60 ⫻ 103/ml), in complete
MCDB medium supplemented with 30% fetal calf serum and 20 ␮g/ml endothelial cell growth supplement. N-MVEC spontaneously formed
anastomosing cords of cells, resembling a capillary plexus, which were well organized by 6 hours (A), and showed a complete honeycomb-like network
after 24 hours (B). When the branching cords of N-MVEC are completely organized, the total number of cells tend to occupy less surface. In the
presence of 250 ng/ml uPA, meshwork formation was increased (C). The process of endothelial cell organization after 6 hours was impaired in the
presence of 1.5 ␮g/ml anti-uPA mAb 5B4 (G) and anti–uPA receptor mAb CD87 (H). Positive control with 10 ng/ml vascular endothelial cell growth
factor (VEGF) (I) is compared with the effect of irrelevant mouse IgG (J). SSc-MVEC failed to form an organized network in Matrigel after 6 hours
(D) and after 24 hours (E). The effect of 250 ng/ml uPA on SSc-MVEC is shown in F. Results are representative of 3 different experiments
performed on 3 normal and 3 SSc cell lines. The values shown are the mean ⫾ SD percentage of the photographic field occupied by cells, and are
reported only for N-MVEC, because SSC-MVEC organization was scarce and irregular. ⴱ ⫽ P ⬍ 0.05 versus control N-MVEC at 6 hours after
plating on Matrigel. (Original magnification ⫻ 100.) See Figure 1 for other definitions.
The mAb against uPA (5B4) and the mAb against uPAR
(anti-CD87) were able to impair the extent of invasion of
normal MVECs and SSc MVECs, whereas irrelevant
mouse IgG had minimal effect. Since the 5B4 mAb
prevents uPA–uPAR interaction, it is likely that in the
spontaneous invasion assay, MVECs exploit the uPAR
association with uPA as it is picked up during the
autocrine process or from the microenvironment. In
turn, the activity of the anti-CD87 mAb, which binds
equally to occupied and unoccupied uPAR, may rely on
impairment of the adhesive interactions of uPAR with
Matrigel-coated ECM macromolecules.
Since the angiogenic microenvironment is rich in
uPA produced by MVECs themselves and by fibroblasts
and macrophages, MVECs were also challenged with
increasing concentrations of uPA (5–500 ng/ml) to stimulate invasion of Matrigel-coated filters (Figure 1B).
The uPA-dependent chemoinvasion was observed to be
3280
dose-dependent, with a maximal effect at 250 ng/ml,
which was much higher for the normal MVECs. The
same mAb that efficiently blocked spontaneous invasion
also impaired uPA-dependent mobilization of MVECs,
whereas irrelevant mouse IgG did not (Figure 1C). The
antibodies had no effect on the viability of normal and
SSc MVECs, as evaluated by trypan blue dye exclusion.
MVEC proliferation. As shown in Figure 1D, uPA
dose-dependently induced proliferation of normal
MVECs and SSc MVECs, with normal MVECs showing
a stronger proliferative response. The same mAb used in
the invasion experiments were able to inhibit uPAdependent proliferation (Figure 1E).
MVEC capillary morphogenesis. The final angiogenic event requires MVECs to organize in interconnecting tubular structures to form the new capillaries. To
study the role of the fibrinolytic system, we undertook
studies of capillary morphogenesis on Matrigel matrices.
Normal MVECs and SSc MVECs were seeded at low
density on plates coated with Matrigel (20,21). In this
assay, MVEC produce elongated processes which eventuate in the formation of anastomosing cords of cells
resembling a capillary plexus (20). The ability to form a
complete network can be sustained by the addition of
angiogenic growth factors to culture medium.
Figure 2A shows that, after 6 hours of plating on
Matrigel, normal MVECs showed abundant networks of
branching cords of cells. By 24 hours, normal MVECs
formed an interconnected network of anastomosing cells
that, by low-power light microscopy, had a honeycomb
appearance (Figure 2B). In comparison, by 6 hours of
assay, SSc MVECs were unable to produce elongated
processes and were unable to organize as branching
cords (Figure 2D). Even after 24 hours (Figure 2E), SSc
MVECs were less prone to spontaneously organize into
a honeycomb morphologic pattern, producing only an
incomplete endothelial network. Although Matrigel is
rich in uPA, the exogenous addition of uPA was able to
augment in vitro morphogenesis by normal MVECs
(Figure 2C) but not by SSc MVECs (Figure 2F).
Figures 2G and H show that antagonism of the
uPA/uPAR system affects capillary morphogenesis.
Normal MVECs were unable to form capillary-like
structures after 6 hours when cultivated in the presence
of the anti-uPA mAb 5B4 (Figure 2G) and anti-uPAR
mAb CD87 (Figure 2H), confirming previous data that
indicate the critical role of uPA, uPAR, and uPA–uPAR
interaction in angiogenesis (10–13). Figures 2I and J
show capillary morphogenesis in the presence of 10
ng/ml VEGF and irrelevant mouse IgG, respectively.
Addition of VEGF to SSc MVECs did not produce any
effect on the organization of the endothelial network
D’ALESSIO ET AL
Figure 3. Surface expression of CD87 and uPA receptor (uPAR)
domain 2 on N-MVEC and SSc-MVEC. A, Dot plots represent the
surface expression of CD87 and uPAR domain 2 in N-MVEC and
SSc-MVEC. B, Histograms represent the surface expression of domain
2 on N-MVEC before and after treatment with 10 nM uPA for 60
minutes. The gate is established using an isotope-matched mAb.
Results in A and B are from 2 distinct experiments. See Figure 1 for
other definitions.
(results not shown). The different angiogenic response
between normal MVECs and SSc MVECs was independent of cell senescence and was observed by performing
experiments with cells from the third to the tenth
passage in culture.
Truncation of uPAR in SSc MVECs. Taken together, our data indicate that the uPA/uPAR system
appears to be impaired in SSc MVECs, in spite of the
presence of a higher amount of endothelial cell–
associated uPAR. We investigated whether such a defect
involved alteration of the uPAR structure in SSc
MVECs such that SSc MVECs would be rendered less
responsive to uPA challenge. RT-PCR showed that
primers that encompass the whole uPAR mRNA yielded
IMPAIRED SSc MVEC ANGIOGENESIS BY MMP-12 CLEAVAGE OF uPAR
amplification products that were indicative of the presence of a complete, nontruncated uPAR in both normal
MVECs and SSc MVECs. This suggests no alteration of
uPAR at the transcriptional level. These data prompted
us to study whether a possible epigenetic uPAR modification could account for the overall decrease of the
uPA/uPAR functions in SSc MVECs.
Figure 3 shows the flow cytometry results in
normal MVECs and SSc MVECs. As shown in Figure
3A, purified normal MVECs and SSc MVECs exhibited
comparable CD87 (uPAR) surface expression when
stained with an anti-uPAR mAb, whereas by using an
mAb specific for the D2 domain of the receptor, the
percentage of cells positive for D2 was higher in SSc
MVECs in comparison with normal MVECs. Treatment
of normal MVECs with 10 nM uPA for 60 minutes,
which has been shown to be able to truncate the receptor
at the D1 level (22), consistently enhanced the percentage of cells stained with anti-D2 mAb (Figure 3B).
These data strongly suggest that anti-D2 mAb recognizes its epitope when D1 is removed. Taken together,
these data support the hypothesis that an active uPAR
cleavage is likely to occur in SSc MVECs.
The ELISA for total uPAR and the D1 domain of
uPAR indicated that D1 was present in almost all of the
uPAR molecules in normal MVECs, whereas it was
detectable in only ⬃40% of the SSc MVEC–associated
uPAR, as shown in Table 1. Parallel experiments were
performed by Western blotting of aliquots of cell lysates
of normal MVECs and SSc MVECs. Figure 4A shows
the immunostaining of uPAR with mAb against D1 and
mAb against D2. The antibody against uPAR D2 revealed 2 bands, both in normal MVECs and in SSc
MVECs, corresponding to the native form (44 kd) and
the truncated form (30 kd) of uPAR. Of note, the
cleaved form was barely detectable in normal MVECs.
The antibody against uPAR D1, which recognizes only
the native form of uPAR, extracted from cell lysates
showed a more intense staining in normal MVECs than
in SSc MVECs, again indicating uPAR truncation in SSc
MVECs. Because of the different uPAR affinity of each
mAb, this differential intensity of immunostaining be-
Table 1. Levels of total uPAR and D1 domain of uPAR in cell
lysates of normal MVECs and SSc MVECs*
Cells
Total uPAR, ng/106 cells
D1, ng/106 cells
Normal MVECs
SSc MVECs
15.3 ⫾ 3.0
23.1 ⫾ 4.3
14.05 ⫾ 3.1
9.33 ⫾ 1.7
* Values are the mean ⫾ SD. uPAR ⫽ urokinase-type plasminogen
activator receptor; MVECs ⫽ microvascular endothelial cells; SSc ⫽
systemic sclerosis.
3281
Figure 4. Cleavage of uPAR and effects of matrix metalloproteinase
12 (MMP-12) in N-MVEC and SSc-MVEC. A, Western blots with
mAb against uPAR domains 1 and 2 were obtained using proteins (50
␮g) from cell lysates loaded in sodium dodecyl sulfate–12% polyacrylamide gels under reducing conditions and blotted to a polyvinylidene
difluoride membrane. Immune complexes were detected with enhanced chemiluminescence. The third lane in the blots for each mAb
shows immunostaining with irrelevant mouse IgG. B, Western blots of
proteins of cell lysates (50 ␮g) of N-MVEC incubated with SScMVEC–conditioned medium were examined under control conditions
(first lane) or in the presence of the indicated protease inhibitors.
Values on the right in A and B indicate the positions of standards with
known molecular weights. ␤-actin was used as a control. C, For
Western blotting with anti–MMP-12 mAb of 20-␮l aliquot of culture
medium, and reverse transcription–polymerase chain reaction (RTPCR) analysis of MMP-12 and GAPDH (control) in N-MVEC and
SSc-MVEC, 5 ␮g of total RNA was reversely transcribed, and reversely transcribed DNA was then amplified with primers. PCR
products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide. Aprot ⫽ aprotinin; Ilom ⫽ ilomastat (see
Figures 1 and 3 for other definitions).
tween the D1 and D2 mAb must not be considered
quantitative.
Overexpression of MMP-12 in cleavage of uPAR
and impairment of capillary morphogenesis in SSc
MVECs. The cleavage of uPAR between D1 and D2 has
been shown to depend on the proteolytic activity of uPA,
directly or indirectly via the plasminogen/plasmin pathway (22,23) or other proteases, such as chymotrypsin
and elastase (24,25). In basic fibroblast growth factor
3282
D’ALESSIO ET AL
Figure 5. Reversal of the antiangiogenic effects of SSc-MVEC–conditioned medium (CM SSc) on N-MVEC by inhibition of MMP-12. A, To assess
invasion of N-MVEC, unstimulated (control) and uPA-stimulated N-MVEC were subjected to Matrigel migration assay as described in Figure 1.
To study the effect of CM SSc on invasion, cells were preincubated 48 hours with CM SSc, and 6.25 ⫻ 103 cells suspended in CM SSc were then
placed in the upper well of the migration chamber. Invasion was allowed to occur for 6 hours following the addition in the lower well of CM SSc
alone or CM SSc supplemented with the indicated inhibitors. Bars show the mean and SD total number of cells/filter after 6 hours. To assess cell
proliferation, 60 ⫻ 103 N-MVEC were seeded in 6 multiwell plates and allowed to grow in MCDB medium supplemented with 30% fetal calf serum
(FCS). After 24 hours, cells were washed and left to grow under the various conditions shown (in 2% FCS). Cells were counted 24 hours later. Bars
show the mean and SD percentage variations of cell number in relation to the number of plated cells. Results are from 3 experiments performed
in triplicate. ⴱ ⫽ P ⬍ 0.05 versus control. B, For capillary morphogenesis assay, N-MVECs were seeded on Matrigel, as described in Figure 2, under
the various indicated conditions. Irrelevant mouse IgG was unable to revert the effect of CM SSc (results not shown). Results were obtained after
6 hours and are representative of 3 different experiments. Values shown indicate the mean ⫾ SD percentage of the photographic field occupied by
cells (original magnification ⫻ 100). See Figures 1 and 4 for other definitions.
(bFGF)/tumor necrosis factor ␣ (TNF␣)– or phorbol
myristate acetate (PMA)–stimulated human MVECs,
truncation of uPAR D1 and D2 has been shown to be
attributable to the hyperactivity of MMP-12 (13). Therefore, we performed a series of experiments to evaluate
the class of protease involved in uPAR cleavage in SSc
MVECs.
Figure 4B shows the results of Western blotting
obtained with the mAb against D1. Twenty-four–hour
incubation of normal MVECs with conditioned medium
containing SSc MVECs strongly reduced D1 expression
in normal MVECs (Figure 4B). A similar reduction in
the levels of D1 in normal MVECs was observed when
SSc-conditioned medium contained a serine-protease
inhibitor, aprotinin, or a cysteine-protease inhibitor,
E64. In contrast, in the presence of a general hydroxamate inhibitor of MMP activity, Ilomastat, D1/D2 uPAR
cleavage did not occur.
These results suggested that the presence of a
member of the MMP family accounted for the truncation of uPAR D1 in SSc MVECs. Therefore, on the basis
of our results and those of previous studies indicating
overexpression of MMP-12 in activated human MVECs
(13), we incubated normal MVECs for 24 hours with SSc
IMPAIRED SSc MVEC ANGIOGENESIS BY MMP-12 CLEAVAGE OF uPAR
MVEC–conditioned medium containing a rabbit mAb
against the hinge region of MMP-12. As shown in Figure
4B, uPAR D1 cleavage was inhibited by this antibody.
RT-PCR analysis showed a significant up-regulation of
MMP-12 mRNA in SSc MVECs (Figure 4C, right), and
Western blot of aliquots of the culture medium indicated
active release of MMP-12 (Figure 4C, left). It is noteworthy that the difference in MMP-12 production between normal MVECs and SSc MVECs is a permanent
alteration, which was observed by using cells from the
third to the tenth passage in culture.
As a consequence of these findings, we performed experiments on uPA-dependent proliferation
and chemoinvasion of normal MVECs preincubated
overnight with SSc MVEC–conditioned medium, and
capillary morphogenesis experiments on normal
MVECs maintained in the presence of SSc MVEC–
conditioned medium. Under these conditions, chemoinvasion and proliferation of normal MVECs (Figure 5A)
were inhibited, and capillary morphogenesis was heavily
impaired (Figure 5B), even in the presence of 250 ng/ml
uPA. The same experiments, performed in the presence
of SSc MVEC–conditioned medium together with Ilomastat or with 10 ␮g/ml anti–MMP-12 antibodies,
showed inhibition of the effects obtained with SSc
MVEC–conditoned medium alone (Figures 5A and B).
DISCUSSION
In this study, we observed that the cell-associated
uPA/uPAR system is hypofunctional in SSc MVECs.
Spontaneous invasion of Matrigel-coated filters as well
as the chemoinvasive and mitogenic effects of uPA were
low for SSc MVECs, whose fibrinolytic pattern was
characterized by higher uPAR expression than that in
normal MVECs. When seeded on Matrigel, normal
MVECs spontaneously formed capillary-like structures,
an activity which was inhibited by uPA and uPAR
antagonism and potentiated by exogenous uPA. SSc
MVECs showed a lower ability to form capillary-like
structures, giving rise to endothelial networks that appeared incomplete, were formed by thinner vessels, and
were not sensitive to exogenous uPA. Taken together,
these data indicate that a lower performance of the
uPA/uPAR system accounts for the decreased ability of
SSc MVECs to perform a complete angiogenic program.
Given the critical role of uPA and uPAR in
angiogenesis (9–13) and the abundance of uPAR in SSc
MVECs, their weak response to exogenous uPA in terms
of invasion, proliferation, and differentiation was difficult to understand, unless uPAR interactions were pre-
3283
vented by a structural alteration. The uPAR has a
3-domain structure: D1 is the N-terminal domain, D2
connects D1 to D3, and D3 is the C-terminal domain
which anchors uPAR to the cell membrane through a
glycosyl phosphatidylinositol tail (26). D1 is involved in
uPA binding and in the interaction with vitronectin (27),
but D2 and D3 are also required for high-affinity
interaction (28). Cell-surface uPAR can be cleaved
within the D1–D2 linker region by several proteolytic
enzymes (22). Such a cleavage leaves, on the cell surface,
a 2-domain uPAR, D2-D3, which is a form expressed in
various tissues and cell lines (22), including human
MVECs (13).
In human MVECs, uPAR D1 is cleaved from the
full-size receptor by MMP-12, following stimulation
either with a combination of bFGF and TNF␣ or with
PMA (13). Truncation of uPAR results in a decrease of
angiogenesis in fibrin matrices (13). We have shown that
in SSc MVECs, uPAR undergoes an MMP-12–
dependent cleavage between D1 and D2, which leaves,
on the cell surface, the D2-D3 form of the receptor
without the ability to bind uPA and to interact with
vitronectin and integrins (29). We have observed that
MMP-12 is constitutively overexpressed in SSc MVECs,
and that uPAR cleavage, impairment of capillary morphogenesis, and reduced uPA-dependent chemoinvasion and proliferation, which were all induced in normal
MVECs by incubation with SSc MVEC–conditioned
medium, could be reversed by coincubation with anti–
MMP-12 antibodies.
The uPAR is a multifunctional protein involved
in the regulation of cell-associated proteolysis, cell adhesion, chemotaxis, migration, and proliferation (30).
These activities of uPAR depend on the presence of the
full-size enzyme, which facilitates its ability to bind uPA
and vitronectin and to interact with integrins. Removal
of uPAR D1 abolishes its interactions with uPA and
vitronectin (28,30) and affects the ability of uPAR to
associate with various ␣- and ␤-integrin chains (29).
Therefore, our observation of an overexpression of
MMP-12 in SSc MVECs, which results in uPAR D1
cleavage, may be critical in the reduced angiogenic
potential of the endothelial cell in the diffuse form of the
disease, by reducing the cell “grip-and-go” properties
related to the full-size form of uPAR (31).
The results described in this report were obtained
with MVECs isolated from biopsy samples of skin, which
were independent from any influence of the microenvironment. Nevertheless, in SSc, MVECs undergo hypoxia
due to subintimal and adventitial fibrosis and intimal
proliferation of arterioles, which leads to a marked
3284
D’ALESSIO ET AL
narrowing of the vessel lumen (32). Many observations
related to tumor angiogenesis indicate that prolonged
hypoxia increases genomic instability (33) and gene
amplification (34), and may act as a selective pressure
for induction of cell variants by altering gene regulation
(35). The endothelium has been considered to be less
likely to develop antiangiogenic resistance, because normal endothelial cells are genetically stable. However,
although endothelial cells have genetic stability and a
unique ability to tolerate hypoxia for prolonged periods,
the situation in SSc may be unique, since MVECs are
subjected to hypoxia for periods ranging from years to
decades, which could determine a unique adaptive response to hypoxic stress.
Perivascular inflammation is a prominent feature
in SSc (36), and the products of inflammatory cells may
induce epigenetic alterations in SSc MVECs. In addition
to producing uPA within the inflammatory microenvironment (37), the activated monocyte/macrophages release both TNF␣ and bFGF (38), which have been
shown to up-regulate MMP-12 (13) and uPAR (39) in
human MVECs. Furthermore, under hypoxia, uPAR
undergoes up-regulation (40). Therefore, we suggest the
possibility that the alterations we have observed in SSc
MVECs (uPAR and MMP-12 up-regulation) may initially be stimulated in an epigenetic manner and then
become the product of the hypoxia-induced selection of
an endothelial cell population more suitable to survive in
the hypoxic microenvironment.
Genetic expression profiling, performed in our
laboratory on normal MVECs and SSc MVECs, indicates that at least 50 genes are differentially expressed in
the 2 cell lines, including uPAR and MMP-12 (D’Alessio
S, et al: unpublished observations). Many of these genes
are related to the onset of angiogenesis and hypoxia,
suggesting that multiple alterations determine the diminished angiogenic capability of SSc MVECs. However, the data reported in the present study indicate that
uPAR cleavage is critical to the pathogenesis of defective angiogenesis, and this a typical feature of the
terminal forms of diffuse SSc.
4.
5.
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7.
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14.
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17.
18.
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