Increased secretion of urokinase-type plasminogen activator by
human lung microvascular endothelial cells
Kimiko
Takahashi1,
Yasuhide
Uwabe2,
Yoshio
Sawasaki3,
Toshio
Kiguchi1,
Hiroyuki
Nakamura1,
Kosuke
Kashiwabara1,
Hisanaga
Yagyu1, and
Takeshi
Matsuoka1
1 Fifth Department of Internal Medicine, Tokyo
Medical College, Ibaraki 300-0395; and 2 Third
Department of Internal Medicine and 3 Department
of Anatomy, National Defense Medical College, Saitama 359-8513, Japan
 |
ABSTRACT |
Human lung microvascular endothelial cells
(HLMECs) secreted 1.5-15 times more urokinase-type plasminogen
activator (uPA) antigen than human hepatic microvascular endothelial
cells, human umbilical vein endothelial cells (HUVECs), angioma
endothelial cells, and lung fibroblasts. All of these cells also
secreted a 100-fold greater amount of plasminogen activator inhibitor-1 than of uPA antigen, and uPA activities were not detected in the culture medium. The expression of uPA mRNA in HLMECs was higher (100-fold) compared with HUVECs, angioma endothelial cells, and lung
fibroblasts. HLMECs secreted uPA antigen on both the luminal and basal
sides of the cells. On the other hand, HLMECs secreted a 10- to 15-fold
lower amount of tissue-type plasminogen activator than HUVECs, mostly
on the luminal side. After stimulation with interleukin (IL)-1
,
HLMECs secreted a six- to ninefold amount of uPA antigen. In contrast,
no stimulatory effect was observed in HUVECs even under high IL-1
concentrations. The secretion of uPA and plasminogen activator
inhibitor-1 from HLMECs was also enhanced by tumor necrosis factor-
and IL-2. These results suggest that HLMECs may contribute not only to
the patency of lung vessels but also to the maintenance of alveolar
functions through the production and secretion of uPA, especially in
the presence of inflammatory cytokines.
tissue-type plasminogen activator; plasminogen activator
inhibitor-1; human umbilical vein endothelial cells
 |
INTRODUCTION |
THE INNER SURFACE OF BLOOD VESSELS is lined with
vascular endothelial cells that differ in morphologies and properties
according to the tissues served by the vessels (21). An examination of such tissue specificity may facilitate our understanding of endothelial functions in different organs. Four major functions of vascular endothelial cells are known: 1) control and transport of
nutritional materials from blood (31); 2) modulation of
vascular tone by producing endothelin, nitric oxide, or prostacyclin
(15, 27, 32); 3) leukocyte transmigration (8); and
4) maintenance of blood fluidity by producing fibrinolytic
enzymes such as plasminogen activators (PAs) (16).
PAs are serine proteases and have been classified into two groups,
urokinase type (uPA) and tissue type (tPA). tPA is strongly connected
with fibrinolysis, and its activity is increased by the coexistence of
a fibrin fragment (25), whereas uPA is related to tissue remodeling
(1). Increased amounts of uPA facilitate uPA turnover, leading to
remodeling of injured tissues including alveoli. Most secreted uPA
binds to the uPA receptor (uPAR) found in the cell membrane of some
types of cells and restricts uPA activity to the cell surface (20). The
bound uPA is believed to activate plasmin, which degrades matrix
components surrounding cells, thereby allowing cell migration. When
uPAR-uPA is inactivated by the binding of PA inhibitor-1 (PAI-1), a
uPAR-uPA-PAI-1 complex is internalized, and uPA-PAI-1 is degraded (17).
The uPAR recycles and becomes available for binding to uPA again (9).
It can be speculated that a large amount of uPA production implies a fast uPA turnover cycle, particularly at sites of tissue injury undergoing repair.
Lavage fluid from normal lung contains a lot of uPA antigen, but the
enzyme activity has not been detected in lavage fluid from patients
with adult respiratory distress syndrome (ARDS), suggesting that uPA
may also serve a protective role in surfactant function (3). Sources of
uPA in the lung have been reported to be macrophages (6) and epithelial
cells (18). Previously, Takahashi et al. (28) reported
that bovine lung microvascular endothelial cells produced and secreted
>10 times as much uPA as bovine aortic, hepatic, and adrenal
microvascular endothelial cells and lung fibroblasts, and uPA antigen
and activity were detected only in those lung cells. Bovine lung
microvascular endothelial cells secreted uPA on both sides of the cell
layer, i.e., on the luminal surface and on the basal surface attached
to basement membrane.
Of course, when the role of human lung diseases is considered, human
lung microvascular endothelial cells (HLMECs) are most appropriate for
study. In a report (7), uPA production has been described as one of the
properties of microvascular endothelial cells derived from human lung.
However, the isolation and culture conditions of human microvascular
endothelial cells are much more complicated than those of the cells
from other species (2, 22). Isolation of human microvascular
endothelial cells has been limited so far. For these reasons,
neither the species nor the tissue specificity of human microvascular
endothelial cells has been fully investigated yet. In this report, we
examined the uPA production of HLMECs and compared the amount with
those of endothelial cells obtained from the other human
tissues. The effect of inflammatory cytokines on the secretion of uPA
and PAI-1 by these cells was also examined.
 |
METHODS |
Materials.
Collagenase, EDTA, sulfuric acid, chloroform, isopropanol, ethanol,
Tris, acetic acid, agarose, and ethidium bromide were from Wako Pure
Chemicals (Tokyo, Japan); dispase was from Godo Shusei (Tokyo, Japan);
newborn calf serum was from Mitsubishi Kasei (Tokyo, Japan); medium
199, keratinocyte-SFM medium, trypsin, TRIzol, Taq polymerase,
2'-deoxynucleoside 5'-triphosphate mixture, DNA ladder,
penicillin-streptomycin mixture, and Fungizone were from
GIBCO BRL (Grand Island, NY); human recombinant basic
fibroblast growth factor was from Intergen (Purchase, NY); human
cellular fibronectin was from Fibrogenex (Chicago, IL); competitive DNA construction kit and competitive RNA transcription kit were from Takara
Biomedicals (Tokyo, Japan); 24-well plates and cell culture inserts
(pore size 0.45 µm) were from Falcon (Lincoln Park, NJ); 60-mm-diameter plastic culture dishes were from Nunc (Naperville, IL);
6-well plates were from Costar (Cambridge, MA);
5(6)-carboxyfluorescein-N-hydroxysuccinimide ester and 96-well
microtiter plates were from Dai-Nippon Pharmaceutical (Osaka, Japan);
anti-human factor VIII complex was from Immunotech (Marseille, France);
avidin-biotin complex staining kit was from Vector Laboratories
(Burlingame, CA); Spectrozyme UK was from American Diagnostica
(Greenwich, CT); uPA ELISA kit was from Monozyme (Hoersholm, Denmark);
PAI-1 and tPA ELISA kits were from Biopool (Uema, Sweden); recombinant
human interleukin (IL)-1
and tumor necrosis factor (TNF)-
were
from Genzyme (Cambridge, MA); and IL-2 was from Takeda Pharmaceutical
(Osaka, Japan). T-primed first-strand kit (Ready-To-Go) was purchased
from Pharmacia (Uppsala, Sweden). Human pro-uPA was obtained from
Abbott Laboratories (Abbott Park, IL) courtesy of Dr. Jack Henkin.
Cell culture.
Small sections of human lung within 1-2 mm of the periphery were
obtained from normal regions of lungs of patients undergoing resection
for solitary lung tumors. The surgery was performed by the Surgical
Service of Kasumigaura Hospital, Tokyo Medical College (Ibaraki,
Japan). HLMECs were isolated according to the modified method
previously described (28). In brief, the sections were digested with
0.1% collagenase, and cells obtained were seeded onto plastic dishes
in growth medium (medium 199 supplemented with 20% newborn calf serum,
10 ng/ml of basic fibroblast growth factor, 100 U/ml of penicillin, and
100 µg/ml of streptomycin) and incubated at 37°C to separate the
endothelial cell-rich suspension from contaminating macrophages. After
1 h, the cells floating in the dish were collected and seeded onto the
fibronectin-coated dish (50 ng/cm2). The lung tissue debris
of the collagenase digestion was further digested with 0.05%
trypsin-0.02% EDTA, and the cells obtained from this digestion were
seeded directly onto a new fibronectin-coated dish. After cell
attachment, the culture was rinsed with PBS, 0.02% EDTA was added to
release nonendothelial cells from the surface of the plates, and,
finally, those nonendothelial cells that were still on the plate were
detached from the surface with a small piece of silicon rubber
connected to a syringe. After the endothelial cell colonies grew large
enough, they were subcultured with a rubber policeman and seeded onto
new fibronectin-coated dishes.
Hepatic tissues were obtained from livers of patients undergoing
resection for solitary liver tumors, and microvascular endothelial cells were cultivated with the same method as the lung microvascular endothelial cells. The angioma endothelial cells were isolated from
venous forearm angioma tissue by treatment with 0.1% collagenase. Human umbilical vein endothelial cells (HUVECs) were cultivated with
500 IU/ml of dispase or 0.1% collagenase with the same method as in a
previous report (29). Lung fibroblasts, which were nonendothelial and
long-shaped cells, were obtained during the preparation of HLMECs. The
culture medium was changed twice a week. Cells in younger generations
(passages 2-7) were used for each examination.
The confluent cell monolayers on the fibronectin-coated 24-well plates
were washed twice with serum-free medium, after which keratinocyte-SFM
containing bovine pituitary extract and epidermal growth factor was
added according to the manufacturer's directions. After 12 h of
incubation at 37°C, the conditioned medium was collected for
determination of uPA and PAI-1. The remaining cells were detached with
0.05% trypsin-0.02% EDTA and counted.
To examine the secretion amount of uPA and PAI-1 from freshly isolated
human lung cells, cells cultivated for 1 wk after the separation were
seeded onto fibronectin-coated 24 well-plates. The 12-h conditioned
medium was collected and uPA and PAI-1 antigens were determined by the
same method described in Measurement of uPA, tPA, and PAI-1
antigens.
Measurement of uPA, tPA, and PAI-1 antigens.
The uPA, tPA, and PAI-1 antigens were determined by conventional ELISA
methods according to the manuals of the kits. In brief, monoclonal
mouse anti-human uPA, tPA, or PAI-1 antibody was coated on 96-well
microtiter plates at 4°C overnight, and samples were added to the
plates to allow binding with the immobilized antibody at 4°C
overnight again. Biotinylated monoclonal mouse anti-human uPA, tPA, or
PAI-1 antibody was added to each well to react with the bound uPA, tPA,
or PAI-1 antigen for 1 h at room temperature, and peroxidase-conjugated
streptavidin was again added to the wells at room temperature. After 1 h of incubation, the contents of the plate were allowed to react for
exactly 30 min with o-phenylenediamine, the substrate for
peroxidase. The plate was measured at 490 nm by a microplate reader
(model 3550-UV, Bio-Rad, Tokyo, Japan) after the reaction was
terminated by the addition of 1 N sulfuric acid. Data are expressed as
means ± SD. Mean values per 104 cells were
determined in triplicate wells for each experiment, and every
experiment was repeated at least three times.
Measurement of uPA activity.
uPA activity in the serum-free conditioned medium was measured
with the chromogenic substrate for uPA, Spectrozyme UK,
after activation of pro-uPA by plasmin treatment as described in a
previous report (28).
Determination of the directionality of uPA and tPA secretion by
HLMECs and HUVECs.
HLMECs or HUVECs were seeded onto the fibronectin-coated membrane (pore
size 0.45 µm) of the cell culture insert for 24-well plates. After
the cells reached confluency, 12-h serum-free conditioned medium using
keratinocyte-SFM was collected from both the upper and lower chambers.
The cells were detached with 0.05% trypsin-0.02% EDTA from each cell
culture insert membrane and counted. The amount of uPA and tPA antigens
in the conditioned medium was measured by the method described in
Measurement of uPA, tPA, and PAI-1 antigens. To confirm that
the cell layer was at confluency, pro-uPA was labeled with fluorescence
and was added to the upper chamber (1 ng/well). After 12 h of
incubation, the fluorescence in the lower chamber was measured with
Fluoroskan II (Dai-Nippon Pharmaceutical) with an excitation wavelength
of 485 nm and an emission wavelength of 538 nm. The confluency of the
cell layer was also confirmed by scanning electron microscopy (30).
RT-PCR.
To obtain total RNA, cells cultivated in 60-mm-diameter dishes were
lysed by the addition of TRIzol. The RNA was extracted with chloroform,
isopropanol, and ethanol by centrifugation after each addition
according to the manufacturer's instructions. The extracted RNA was
dissolved in distilled water, and the absorbance was measured with a
spectrophotometer (UV-1600, Shimazu, Kyoto, Japan) at 260 nm to
estimate the amount of total RNA. To prepare template cDNA, equal
amounts of total RNA were added to tubes of a T-primed first-strand kit
including Not I-(dT)18 primer, 2'-deoxynucleoside 5'-triphosphates, and RT, and the tubes
were shaken at 37°C. After 60 min of incubation, uPA sequences
were subsequently amplified by PCR with 1 µl of cDNA template, 0.5 U/ml of Taq polymerase, and 1 µM sense and antisense oligomer primers in a total volume of 10 µl. The set of primers specific for
human pro-uPA from a transformed human endothelial cell line (29, 33),
from which an amplified fragment of 459 bp was obtained, consisted of a
forward primer beginning at bp 769 with a nucleotide sequence of
5'-GCCTTGCTGAAGATCCGTTCCAAGGAGGGC-3' and a reverse primer beginning at
bp 1198 with a nucleotide sequence of
5'-CAGGCCATTCTCTTCCTTGGTGTGACTGCG-3'. RT-PCR was carried out under the
following conditions: 1 cycle at 95.0°C for 3 min; 30 cycles at
95.0°C for 45 s, 65.0°C for 45 s, and 72.0°C for 3 min; and 1 cycle at 72.0°C for 5 min (Program Temp Control System PC-800, ASTEC,
Fukuoka, Japan). The PCR reaction products (3-7 µl) were
separated by electrophoresis on a 2.0% agarose-Tris-acetate-EDTA gel
and stained with ethidium bromide.
For quantitative analysis of uPA mRNA in human endothelial cells, a
competitive RT-PCR method was used (13). The 383-bp competitor DNA for
uPA that has the sense and antisense uPA primer sequences at its both
ends was made with a competitive DNA construction kit using
DNA as a
template. The competitor RNA was also made with a competitive RNA
transcription kit using the competitor DNA according to the
manufacturer's manual. The competitor RNA (9.6 × 105 to 9.6 × 107 copies)
was added to each total RNA (1.56 ng) sample from the cultured
endothelial cells, and RT-PCR was carried out with the same procedures
as described above.
Cytokine treatment.
After the monolayers of human lung endothelial cells were treated with
one of the inflammatory cytokines (IL-1
, 1 ng/ml; IL-2, 0.1 ng/ml;
or TNF-
, 1 ng/ml) for 10 h, they were maintained in serum-free
medium (without the cytokines) for 12 more h and collected, and the
cell number in each well was counted. The amount of uPA and PAI-1
antigens in the conditioned medium was measured as described in
Measurement of uPA, tPA, and PAI-1 antigens. In separate
examinations, 0.1, 1.0, or 10.0 ng/ml of IL-1
were added to the
cultures of HLMECs and HUVECs. After 12 h of incubation, the
conditioned medium was collected, and the amount of uPA antigen was
measured. Data are expressed as mean ± SD. The mean values per
104 cells were determined in triplicate or duplicate for
each experiment. For statistical analysis, Student's t-test
was used.
 |
RESULTS |
The confluent monolayer of isolated pure cultures of HLMECs showed a
typical cobblestone monolayer, and the representative endothelial
marker, von Willebrand factor, was detected as in other endothelial
cells but not in fibroblasts (data not shown). With the use of these
cells, the amount of uPA and PAI-1 antigens secreted into the culture
medium was determined. The amount of uPA antigen detected in HLMEC
medium was significantly higher ( > 1.2-1.9 times) than in
the medium of hepatic microvascular endothelial cells (P < 0.05), angioma endothelial cells (P < 0.001), HUVECs
(P < 0.001), and lung fibroblasts
(P < 0.001; Fig.
1A). The amount of PAI-1 antigen
secreted from the HLMECs was significantly higher than that secreted by
the hepatic microvascular (P < 0.01) and angioma
endothelial cells (P < 0.05) but lower than that
secreted by HUVECs (P < 0.01) and lung fibroblasts
(P < 0.05; Fig. 1B). In all cases, the amount
of PAI-1 antigens secreted exceeded the amount of uPA secreted by
~100-fold. In fact, uPA activity was not detected at all in the
conditioned medium when measurements were made with chromogenic
substrate (data not shown). The freshly isolated human lung endothelial
cells (the purity of endothelial cells was > 90%) secreted
comparable amounts of uPA and PAI-1 as the purified and serially
passaged HLMECs (Fig. 2).

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Fig. 1.
Amount of urokinase-type plasmogen activator (uPA; A) and
plasminogen activator inhibitor-1 (PAI-1; B) antigens
secreted from human lung microvascular endothelial cells (HLMECs),
liver microvascular endothelial cells, angioma endothelial cells, human
umbilical vein endothelial cells (HUVECs) and fibroblasts. Confluent
cultures of each cell type were washed and added to serum-free medium.
After 12 h of incubation, conditioned medium was collected, and uPA and
PAI-1 antigens were measured by ELISA method. Values are
means ± SD; n, no. of samples. P values
compared amount of antigens secreted by HLMECs.
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Fig. 2.
Amount of uPA (A) and PAI-1 (B) antigens secreted
from freshly isolated human lung cells. One-week-old cultured human
lung cells were inoculated onto fibronectin-coated 24-well plates and
added to serum-free medium. After 12 h of incubation, conditioned
medium was collected, and uPA and PAI-1 antigens were measured by ELISA
method. Freshly isolated human lung cells were not pure microvascular
endothelial cells. Because they also included alveolar epithelial cells
and fibroblasts, experiments were carried out 3 times (nos. on
x-axis). Values are means ± SD.
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To examine uPA mRNA levels, total RNA fractions were obtained from
various endothelial cells and lung fibroblasts and analyzed by the
RT-PCR method described in METHODS. A difference in the expression of uPA mRNA was observed among the cells. The same result
was observed in all five experiments. uPA mRNA was clearly detected in
unstimulated HLMECs and hepatic microvascular endothelial cells, but
the expression level was much lower in HUVECs and lung fibroblasts
(Fig. 3A). The estimated amount
of uPA mRNA in the RNA preparation from HLMECs (1.56 ng as total RNA)
was 9.6 × 107 copies but was
9.6 × 105 copies in that from HUVECs by competitive
RT-PCR (Fig. 3B). The uPA mRNA in liver microvascular
endothelial cells was < 9.6 × 105 copies
and in angioma cells and fibroblasts was not detected in these
experiments.

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Fig. 3.
Detection of uPA mRNA in HLMECs, liver microvascular endothelial cells,
angioma endothelial cells (Ang.), HUVECs, and lung fibroblasts (Fib.).
RT-PCR was performed on total RNA as described in METHODS.
A: a single band is seen in each lane, which has an identical
no. of base pairs (459 bp) as the predicted size of
fragment given by primers specific to uPA mRNA. HUVECs express about
one-half the amount of amplified uPA mRNA as HLMECs as determined by
image analyzer. B: competitive RT-PCR was also performed on
coexistence of competitor RNA for uPA (383 bp) with
9.6 × 105 (lanes 1, 4, and 7),
9.6 × 106 (lanes 2, 5, and 8), and
9.6 × 107 (lanes 3, 6, and 9)
copies of added competitor. Lanes 1-3: HLMECs; lanes
4-6: human liver endothelial cells; lanes 7-9:
HUVECs.
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For determination of possible direction specificity in uPA secretion,
HLMECs were cultured on the fibronectin-coated polystyrene membrane of
cell culture inserts. The cells expressed as a confluent monolayer on
the membrane (Fig. 4). After 12 h of
incubation in serum-free conditioned medium, the conditioned medium was
collected from both the upper and lower chambers. uPA antigens were
found in both chambers (Fig. 5A).
To examine whether the secreted uPA to the upper chamber passed through
the endothelial cell layer or whether the cells secreted uPA in both
directions after the HLMECs reached confluency on the membrane of the
cell culture insert, an excess amount of fluorescence-labeled
single-chain uPA was added to the upper chamber. Neither the
fluorescence nor the increase in uPA antigen in the lower chamber was
detected after 12 h of incubation (data not shown). On the other hand, tPA antigen was found mainly in the upper chamber (100% HUVECs; 85%
HLMECs; Fig. 5B).

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Fig. 4.
Phase-contrast microphotograph of confluent monolayer of HLMECs on
membrane of cell culture insert. Magnification, × 500.
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Fig. 5.
Directionality of uPA (A) and tissue-type plasminogen
activator (tPA; B) secretion by HLMECs and HUVECs. Confluent
monolayers of HLMECs or HUVECs on membranes of cell culture inserts
were fed with serum-free medium. After 12 h, amount of uPA and tPA
antigens in upper and lower chambers was measured. Values are
means ± SD; n = 10 samples for uPA and 3 samples for
tPA.
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Alteration in the uPA amount secreted from both HLMECs and HUVECs was
measured after treatment with different concentrations of IL-1
.
After 12 h of stimulation with IL-1
, the amount of uPA antigen
secreted by HLMECs increased in a dose-dependent manner, and the
increase reached almost sixfold with the concentration as low as 0.1 ng/ml (Fig. 6A). In contrast, the
amount of uPA antigen in HUVEC-conditioned medium was unaffected by
IL-1
treatment. On the other hand, a 10- to 15-fold amount of tPA
was detected in HUVEC-conditioned medium compared with that in HLMECs
(Fig. 6B). But the amount of tPA secretion did not respond to
the addition of IL-1
. The confluent monolayers of HLMECs were
treated with low levels of IL-1
, TNF-
, or IL-2 for 10 h. The
three cytokines stimulated significant (6- to 9-fold) increases in the
secretion of uPA antigens by HLMECs (IL-1
and IL-2,
P < 0.001; TNF-
, P < 0.01; Fig.
7A). The amount of PAI-1 secreted
by HLMECs was also significantly increased by treatment with IL-1
and TNF-
(P < 0.01; Fig. 7B). No
significant changes in cell number or morphology were observed after
the treatments.

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Fig. 6.
Dose response of uPA (A) and tPA (B) secretion by
HLMECs ( ) and HUVECs ( ) treated with
interleukin (IL)-1 in serum-free keratinocyte-SFM. After 12 h of
incubation, culture medium was collected, and amount of uPA and tPA
antigens was measured. Values are means ± SD; n = 6 samples.
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Fig. 7.
Effect of cytokines on secretion of uPA (A) and PAI-1
(B) by HLMECs. Cells were treated with 1 ng/ml of IL-1 ,
0.1 ng/ml of IL-2, or 1 ng/ml of tumor necrosis factor (TNF)- for 10 h, and medium was changed to serum-free keratinocyte-SFM. After an
additional 12 h of incubation, medium was collected, and cells were
counted. Amount of uPA and PAI-1 antigens in conditioned medium was
measured by ELISA method. Values are means ± SD; n, no.
of samples. P values compared amount of antigens secreted by
cytokine-untreated HLMECs.
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 |
DISCUSSION |
The results of the present study clearly show that uPA is actively
produced and secreted by microvascular endothelial cells from human
lung and liver, but endothelial cells from large vessels produced less
uPA (Figs. 1A and 3). Although the freshly isolated human lung
endothelial cells were not pure endothelial cells (purity ~90%),
they secreted comparable amounts of uPA and PAI-1 as the purified and
serially passaged HLMECs (Fig. 2). We also found that there was a
distinct difference between HLMECs and HUVECs on the reactivity of uPA
and tPA production when stimulated by IL-1
(Fig. 6). It has been
reported that HUVECs in primary culture produce almost exclusively tPA
but progressively produce uPA after multiple passages (5). In the
present experiments, endothelial cells (including HUVECs) were in
younger generations (passages 2-7). Therefore, we
expected to find little or no uPA activity in the HUVEC-conditioned
medium. HLMECs secreted lower amounts of tPA than HUVECs. It suggested
that uPA may work in a different way from tPA in vivo.
Many reports have demonstrated the heterogeneity of endothelial cells
(21), especially with regard to angiogenesis (34), vulnerability to
pathological conditions (26), and the distribution of membrane proteins
(10). Interestingly, although a clear difference was found in the
amount of uPA antigen, such a tissue specificity was not so obvious
from the results of RT-PCR experiments, and slight but not negligible
bands of uPA mRNA were detected in HUVECs (Fig. 3). In competitive
RT-PCR, a semiquantitative RT-PCR method, the uPA mRNA expression in
HLMECs was 100-fold higher than that in HUVECs. This result was the
reflection of the ELISA data (Fig. 1). But there was less expression of
uPA mRNA than the level assumed from the ELISA value in human liver
microvascular endothelial cells. These discrepancies might reflect a
difference in the turnover of mRNA, the posttranscriptional modulation
such as processing or translation rate, the size of membrane and
intracellular uPA pools, or the secretion rate of uPA.
All cells examined here also secreted about a 100-fold higher amount of
PAI-1 than of uPA (Fig. 1B); the amount of uPA activated may
be regulated by inhibitors to ensure that the quantity of tissues or
proteins degraded is not excessive (4). Almost all uPA activity on the
cell surface and in the culture medium might bind PAI-1 and be
inactivated. Takahashi et al. (28) reported that bovine lung
endothelial cells secreted almost negligible antigens of PA inhibitors.
The differences could be construed as evidence for species specificity
(11), which emphasizes the importance of the study using human cells
for the interpretation of clinical significance, indicating that the
use of animal cells may be misleading.
As in bovine cells (28), human lung endothelial cells secreted uPA
antigen not only from the luminal surface but also from the surface
attached to the basement membrane (Fig. 5A). Spontaneous leakage of uPA, which was secreted into the upper chamber, was not
present when the fluorescence-labeled uPA was added to the upper
chamber (data not shown). The basement membrane of lung microvascular
endothelial cells is in immediate contact with alveolar epithelial
cells. uPA antigen was detected on the normal alveolar surface (3), and
macrophages (6) or epithelial cells (18) have been suggested as a
possible source of uPA. From the present findings, it seems likely that
HLMECs may also supply uPA to alveolar surfaces through basement
membrane-directed secretion as do bovine lung microvascular endothelial
cells (28). It has been postulated that fibrinogen or other plasma
proteins that leak into the alveolar spaces and interfere with the
function of surfactant may normally be removed by uPA. In contrast, tPA
from both HLMECs and HUVECs was secreted into the upper chamber (Fig.
5B). It suggests that the physical role of uPA may be
different from that of tPA.
Secretion of uPA from HLMECs was potentiated with IL-1
, whereas no
stimulation was found in HUVECs even under high IL-1
concentrations
(Fig. 6A). In contrast, the secreted amount of tPA from
HLMECs and HUVECs was not changed by the IL-1
concentration (Fig.
6B). The different response to this cytokine may also
represent the tissue specificity of endothelial cells. Other cytokines, TNF-
and IL-2, also increased the secretion of uPA and PAI-1 from
HLMECs (Fig. 7). In these experiments, the exposure to cytokines was
brief, and the concentration of cytokines used in these experiments was
much lower than that inducing morphological change or detachment of the
cells (19, 23). Kiguchi et al. (14) reported that such minimal exposure
to cytokines induced intercellular adhesion molecule-1 expression in
bovine lung microvascular endothelial cells, which was potentiated by
subthreshold hypoxia. IL-1
and TNF-
are known to be important
inflammatory cytokines and coexist at sites of inflammation (23).
Tissue-constructing cells at inflammatory sites are often damaged and
need repair (19, 23). The actions of these cytokines on endothelial
cells that promote leukocyte adhesion and activation are likely to be
important in the development of acute inflammatory responses (14, 23). After the adhesion of leukocytes to endothelial cells, the leukocytes migrate into the surrounding tissues to support tissue repair (23). For
this migration and the subsequent angiogenesis as a tissue repair
mechanism, uPA is a very important protease involved in the degradation
of connective tissues (12). The increased secretion of uPA and PAI-1 by
inflammatory cytokines might play a significant role in acute and
chronic lung inflammatory diseases. For example, uPA secreted from
HLMECs might be directed to the cells that express uPAR, including
endothelial cells themselves and macrophages. The uPA-uPAR binding
would induce the migration of these cells and might help tissue repair
at the inflamed sites. This postulate is based on the findings that
ARDS is characterized by morphological and biological damage to lung
microvascular endothelial cells. These cells provide protection against
the development of acute lung injury and also participate in the repair
of alveolar tissue after injury (3, 12, 24). However, further detailed examinations are needed before we can discuss a possible relationship between the present results and the pathogenesis of lung inflammation.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Katsueki Watanabe (Tokyo Medical College, Tokyo,
Japan) for providing the angioma tissue and Drs. Kazuo Yoneyama and
Bor-Rong Wei (Kasumigaura Hospital, Tokyo Medical College) for
providing lung tissues. We also thank Masako Mitsui for excellent technical assistance.
 |
FOOTNOTES |
Address for reprint requests: K. Takahashi, Department of Internal
Medicine, Tokyo Medical College, Kasumigaura Hospital, 3-20-1, Chuou,
Ami, Inashiki, Ibaraki 300-03 Japan.
Received 24 April 1997; accepted in final form 27 March 1998.
 |
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