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INTRODUCTION |
Shear stress, elicited by blood flow, represents an important
signal for endothelial cells to synthesize vasoactive autacoids. In
addition to an acute increased autacoid production there are also
chronic effects of shear stress. One such effect is the differential expression of some proteins in endothelial cells (1, 2). It has been
shown by several groups that, in particular, the expression of the
endothelial nitric-oxide synthase (eNOS)1
is increased when endothelial cells are exposed to laminar shear stress
for several hours (3-10). To date, our knowledge of the exact signal
transduction cascade from the stimulus (shear stress) to the effect
(gene expression) still remains incomplete. In particular, little is
known about the role of the extracellular matrix in this signaling
process, although several lines of evidence support a role for the
cell-matrix interaction in shear stress-induced cell activation
(11-14).
Several authors have reported an alignment of endothelial cells in the
direction of the shear force applied to cultured cells and blood
vessels (13-18). Such a reorientation must involve the formation of
new cell-matrix contacts. Indeed, focal adhesion points change
dynamically in endothelial cells under shear stress (19). These focal
adhesion points represent not only the connection between matrix and
cell membrane but also are colocated in a number of regulated kinase
activities in these sites, which may be involved in the translation and
transduction of extracellular signals across the cell membrane (20). If
such focal adhesion complexes are active as a link between the shear
stress and subsequent gene expression, the protein composition of the
extracellular matrix should play a very important role.
Among these matrix proteins, laminins represent the major glycoprotein
family in all basement membranes and as such are of particular
interest. In addition, the cellular laminin secretion under shear
stress appears to be up-regulated, whereas fibronectin seems to be
down-regulated (21-23). Previous studies have shown that members of
the laminin family specifically promote cell growth, differentiation,
and migration (24). Data support that part of these effects are
dependent on a so-called "laminin-binding protein" (LBP) of 67 kDa
in size (25). This 67-kDa LBP was identified to bind highly selectively
to a certain sequence of the laminin
1 chain, namely
YIGSR, and to promote cell attachment as well as migration (26,
27).
In this study laminin I was examined for its potential role in the
shear stress-mediated expression of the eNOS. Special attention was
given to the laminin-cell interaction via the non-integrin 67-kDa LBP
and its involvement in the signal transduction of shear stress-mediated
eNOS expression.
There is ample literature about the 67-kDa LBP involvement at the
pathophysiologic level, such as tumor invasion; however, there is
little mention of a physiologic role for the LBP. It was our reasoning
that the function of the 67-kDa LBP in the above mentioned signal
cascade downstream of the shear stress could represent a major
physiological task of this LBP in the vascular system.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
From fresh porcine aorta obtained from the
local slaughterhouse, endothelial cells were isolated following
standard procedures (28). Briefly, the fat and connective tissue were
trimmed from the aorta with sterile scalpels, and the vessel was cut
longitudinally. After washing with sterile phosphate-buffered saline
(PBS), vessels were put into a frame with endothelial side facing up.
At this point they were incubated with dispase (Boehringer Mannheim;
2.4 units/ml PBS) for 20 min at 37 °C in a humidified incubator.
Endothelial cells were dislodged with sterile medium (Dulbecco's
modified Eagle's and Ham's F-12 media, 1:1), collected, and
thereafter seeded onto standard plastic culture dishes in Dulbecco's
modified Eagle's and Ham's F-12 media (1:1) containing
penicillin/streptomycin, glutamine (200 mM), and 20% fetal
calf serum (Biomol). The cell culture medium was replaced every other
day. Only cells from passage 1 were used for shear stress experiments,
and the corresponding control cells were obtained from identical pools.
Coating of Glass Plates, Application of Shear Stress and
Inhibitory Peptides--
Plastic culture dishes were not suitable for
the shear stress experiments because they are not perfectly planar, and
they displayed a high degree of variability as to the bottom thickness. Therefore glass plates were used to create a monolayer of endothelial cells for the application of shear stress. Before inoculation with the
cells, the plates were coated following standard procedures. Briefly,
clean glass plates were each incubated with 20 µg/ml laminin I (from
Engelbreth-Holm-Swarm tumor, purchased from Sigma), laminin fragments
P1 and E8 (a generous gift from Lydia Sorokin, Erlangen), collagen I
(from Boehringer), fibronectin (human sera purchased from Boehringer),
or they were used without any coating. The endothelial cells were
allowed to adhere and were cultivated up to confluence (approximately 3 days). The glass plates were then placed into a rotating cone shear
apparatus. For a glass plate with a diameter of 10 cm, an angle of 1°
of the cone guaranteed a homogeneous laminar shear stress. The rotating
speed was adjusted in a way so that the shear stress generated was 16 dyn/cm2. Incubations were performed in complete culturing
medium with reduced serum content (1% fetal calf serum for 6 h).
Attachment Experiments--
Attachment experiments were done
following the paper of Iwamoto et al. (26). At the time of
seeding, 150 µg/ml of the YIGSR peptide or YIGSK for control was
added. After various intervals, cellular attachment was recorded
microscopically by counting cells in 10 randomly distributed optical fields.
Acute effects of YIGSR on attachment, in the presence of shear stress,
were tested as follows. Endothelial cells were allowed to adhere to
laminin I for 16 h, and the peptide YIGSR (150 µg/ml) was added
just before the shear stress. All attachment experiments were done in triplicate.
To test the effects on shear stress-mediated signal transduction via
the 67-kDa LBP, the inhibitor-peptide YIGSR against LBP as well as the
control peptide YIGSK were added at a much lower concentration (35 µg/ml each), 3 days after initial seeding and immediately before the
onset of shear stress.
Northern Blots--
After applying shear stress to the porcine
aortic endothelial cells the cell medium was aspirated. To the cell
layer 2 ml of Trizol reagent (Life Technologies, Inc.) for each 10-cm
dish was added, and total RNA was isolated following the instruction of
the company. 10-20 µg of total RNA was denatured and loaded onto a
formaldehyde/formamide-agarose gel. After completion of the
electrophoresis, the RNA was transferred to nylon membranes (Nytran,
Schleicher & Schuell) with capillary blotting. After fixation with UV
light (320 nm, 0.7 J/cm2) the membranes were blocked with
4 × SSPE, 50% formamide, 4 × Denhardt's solution, 0.1%
SDS, 100 ng/ml salmon testis DNA for 2-3 h at 42 °C. A randomly
[32P]dCTP-labeled cDNA probe for eNOS (base pairs
3107 to 3405) was added with fresh buffer and incubated for 24 h
at 42 °C. After washing twice for 15 min at room temperature in
2 × SSC and 0.1% SDS and once for 15 min at 65 °C in 1 × SSC and 0.1% SDS, the filter was exposed to autoradiographic films
(NEF 490, DuPont) up to 5 days at
80 °C.
To monitor differences in the amount of RNA loaded, the filter was
reprobed with a full-length cDNA probe for
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Stripping was
performed according to Schleicher & Schuell. Briefly, the membrane was
incubated for 30 min in 50% formamide and 6 × SSPE (sodium
chloride/sodium phosphate/EDTA) at 65 °C. The hybridization and
washing protocol was the same as for eNOS.
The films were scanned with a videodensitometric system (Bio-Rad), and
band intensities for eNOS were normalized to their corresponding GAPDH
bands. Finally, this ratio for the cells under shear stress was
compared with its control under static incubation.
Semiquantitative Reverse Transcriptase-PCR--
Alternatively to
Northern blots, the eNOS mRNA expression was assessed by
semiquantitative reverse transcriptase-PCR. The extraction of total RNA
was the same as for Northern blots. The primers used were
5'-CCTTCCAGGCCTCCTGTGAGACTT-' (2116-2140) and 5'-ACGTGGAGCAGA-CTCCATAGTGCA-3' (2948-2924) from porcine eNOS mRNA. Following the protocol of the manufacturer (Titan kit from Boehringer), all necessary components except the enzyme mix and the
specific primers were pipetted together in a 2 × setup. At this
point the mix was divided into two halves, and the primers for eNOS and
GAPDH were added. Loading discrepancies were thus reduced to a minimum.
The number of cycles in which the PCR was still in the linear range was
tested out in previous experiments, and the relative expression of eNOS
mRNA was calculated according to the Northern blot experiments by
normalization of eNOS bands to their corresponding GAPDH bands.
Western Blots--
For protein analysis of eNOS protein,
endothelial cells were dislodged through incubations in 15 mM sodium citrate and 135 mM KCl, pH 7.0, on
ice for 30 min. The cell pellets were resuspended in lysis buffer (20 mM KH2PO4, pH 7.0, 1 mM
EDTA, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin)
and disrupted by passing 20 times through a 26-gauge injection needle.
Total protein content was estimated by Bradford protein analysis.
Cleared lysates (20 µg of total protein) were separated on a 10%
polyacrylamide and SDS gel following standard procedures. After
separation, proteins were transferred to nitrocellulose membranes
(Schleicher & Schuell) in a semidry blotting apparatus, using 39 mM glycine, 48 mM Tris base,
0.037% SDS, and 10% methanol as the transfer buffer and 0.8 mA/cm2 for 1 h as the power setting. After finishing
the transfer, the membranes were stained briefly with Ponceau S to
monitor equal loadings and blocked with 5% dry skim milk powder in PBS
for at least 4 h. The first (specific) antibody, either anti-LBP,
which was a generous gift from Hynda Kleinman (NIH, Bethesda) or
anti-eNOS, which was purchased from Dianova, was incubated in 5% milk
powder in PBS for 1 h at room temperature or overnight at 4 °C.
After four washings at room temperature in 0.1% Tween 20 and PBS, the second antibody (Sigma) was incubated in 5% milk powder in PBS for 30 min at room temperature. After four washes in 0.1% Tween 20 and PBS, a
second antibody detection was performed either with chemiluminescence
for horseradish peroxidase following the instructions to the Boehringer
kit used or with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate staining for alkaline phosphatase. Detected bands were recorded and quantified with a videodensitometric system from Bio-Rad.
For characterization of matrix proteins endothelial cells were
dislodged from the plates nonenzymatically by incubation with 15 mM sodium citrate and 135 mM KCl, pH 7.0. Cells
were scraped off the matrix, and the plates were washed three times
with PBS (free of magnesium and calcium) for 5 min each. Subsequently
the matrix proteins were removed with boiling SDS buffer (10 mM Tris, pH 6.8, 1% SDS). The protein concentration was
measured according to Zaman and Verwilghen (29). Equal amounts of total
matrix protein were loaded onto a 10% polyacrylamide gel, and gel
electrophoresis was performed as described for the eNOS-Western. The
specific first antibody serum (a generous gift from Lydia Sorokin
(Erlangen) for laminin
1,
1,
1 and
fibronectin) was used in 3% skim milk and PBS.
Nitrite/Nitrate Measurements--
Estimations of the
NOX as the sum of nitrite and nitrate produced
by endothelial cells during shear stress were performed after the
reduction of nitrate to nitrite with cadmium. Briefly, cells were
washed before the experiments with warm PBS (supplemented with calcium
and magnesium) and finally incubated with fresh cell culture medium.
After 6 shear stress the supernatant was collected from the shear
stress as well as the control cells. Duplicate aliquots of 100 µl
were deproteinized with ZnSO4 and incubated with cadmium
pellets. After 16 h the samples were mixed with p -aminobenzenesulfonamide and
N-(1-naphthyl)ethylenediaminedihydrochloride reagent, and
the optical density was read in a spectrophotometer at 540 nm. The
calibration curve was constructed with nitrite as well as nitrate
standards. The calculated concentrations of NOX
were normalized to mg of total cellular protein and are expressed as
nmol/h. For laminin I, 11 experiments were performed.
Statistical Analysis--
All shear stress experiments were
performed with paired cultures from the same preparation lot.
Student's t test for paired experiments was used to
evaluate differences in the results. An error level of
p < 0.05 was considered significant. The number of
experiments done are indicated as n. Error bars
represent the S.E.
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RESULTS |
Analysis of Matrix Protein Composition--
Even after 3-5 days,
which the endothelial cells needed to reach confluence, there was a
clear distinction with regard to the matrix composition of cells grown
on laminin I, collagen I, fibronectin, or glass alone.
Although the macroscopic inspection of the protein composition of the
matrix indicated no differences (see Coomassie-stained gel in Fig.
1), no laminin I could be detected on
collagen I, on fibronectin-precoated or on pure glass plates, whereas
on laminin I-coated plates a clear laminin I signal was obtained (see
Fig. 1C). Fibronectin, however, was found in each matrix
composition regardless of the previous coating procedure.

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Fig. 1.
Western blots for the matrix proteins laminin
and fibronectin. Porcine aortic endothelial cells were grown under
static conditions with different precoatings (1,
fibronectin; 2, collagen I; 3, pure glass; and
4, laminin I). Matrix proteins were collected
nonenzymatically from these culture dishes. Equal amounts were
separated on a reducing SDS-polyacrylamide gel and blotted onto
nitrocellulose. Panel A represents a gel stained with
Coomassie Blue for total protein. Panel B is a Western blot
probed with an antibody against fibronectin and panel C,
against laminin I.
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In contrast to the findings above, when plastic dishes were used
instead of glass plates, we always found laminin I in the matrix,
regardless of the initial coating with fibronectin, collagen I, or
laminin I (data not shown).
Expression Analysis of eNOS--
Alterations in the expression
level of the eNOS mRNA were analyzed with Northern blots as well
with reverse transcriptase-PCR. Fig.
2A shows a typical Northern
blot. The upper row represents the hybridization signal with
the eNOS probe, whereas the lower row shows the one for
GAPDH.

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Fig. 2.
Northern blot for eNOS mRNA.
Panel A, RNA from porcine aortic endothelial cells, grown on
pure glass or on the different precoatings with laminin I, fibronectin,
or collagen I, was isolated after a 6-h shear stress of 16 dyn/cm2. 10-20 µg of total RNA was separated on a
formaldehyde/formamide agarose gel. In a first step the blot was probed
for eNOS mRNA (upper row), and then the filter was
reprobed for GAPDH (lower row). S represents the
shear stress-treated cells, and C represents the static
control from the corresponding cell pool. Panel B,
videodensitometric quantification of several Northern blots was
analyzed as follows. The densities of specific eNOS mRNA bands of
all experiments were related to their corresponding GAPDH bands. The
relative expression represents the eNOS mRNA of the shear stress
experiments normalized to their static controls, which was set at 1 (fibronectin, n = 9; glass, n = 13;
collagen I, n = 9; laminin I, n = 16).
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Note that for laminin I coating, the cells showed an apparent increase
of eNOS mRNA. No obvious change in the eNOS expression was found in
the case of coatings with collagen I and fibronectin as well as without
any coating. The GAPDH expression showed no alterations caused by shear
stress or coating procedure.
In Fig. 2B the results of different experiments are
summarized. The relative expression of eNOS in endothelial cells after a 6-h shear stress (16 dyn/cm2) was not significantly
altered in the case of coating with fibronectin (1.1-fold over static
control, n = 9) or in cells grown directly on glass
(1.4-fold over static control, n = 13). Only a moderate increase could be detected for cells grown on collagen I-coated glass
plates (1.4-fold over static control, p < 0.05, n = 9).
In contrast to the above findings, a highly significant
(p < 0.001, n = 16) 2-fold increase in
eNOS expression was detected in the case of laminin I-coated glass
plates. This elevation of eNOS mRNA expression was caused by new
transcription because repeating the experiments in the presence of
actinomycin D (5 µg/ml) prevented an increase of specific mRNA
for both GAPDH and eNOS (data not shown). The addition of recombinant
laminin I (from Engelbreth-Holm-Swarm tumor) to the superfusate of
static control cells had no stimulating effect on the eNOS expression
(data not shown).
Western Blot Analysis of eNOS Protein--
The Western blots
performed showed that the eNOS mRNA elevation is not only transient
without effects on translation. Fig. 3A represents a typical
Western blot for cells grown on a laminin matrix; in Fig. 3B
Western blots are shown for coatings with fibronectin, collagen I, and
uncoated glass, all showing a single band at 140 kDa. In Fig. 3,
C and D several experiments are summarized. After a 6-h shear stress treatment, an increase in eNOS protein of 1.9-fold over control (n = 4, p = 0.054) was
observed only for laminin.

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Fig. 3.
Western blot for eNOS protein.
Panel A, a representative Western blot is shown of total
cell lysates from porcine aortic endothelial cells grown on laminin
I-precoated plates either under static control conditions or under a
16-dyn/cm2 shear stress for 6 h. Panel B,
representative Western blots of total cell lysates from endothelial
cells grown on fibronectin, pure glass, or collagen I, which were
subjected to 16 dyn/cm2 for 6 h. All experiments were
quantified with a videodensitometer and normalized to the static
control, which was set at 1. Panel C, laminin I coating
(n = 4, p = 0.054); panel D,
collagen I, fibronectin, and pure glass (n = 2 each).
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Measurements of NOX Production under Shear
Stress--
As shown in Fig. 4, the
NOX production as derived from nitrite/nitrate
release into the supernatant medium was found to be increased after
shear stress. After 6 h in control cells, 27.2 nmol/mg of
protein/h NOX was measured, which was elevated
up to 42.1 nmol/mg of protein/h under shear stress.

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Fig. 4.
Nitrate production under shear stress.
Porcine aortic endothelial cells were grown on a laminin matrix and
exposed to a 16-dyn/cm2 shear stress. After 6 h the
experiments were stopped, and nitrate in the supernatant was measured
with the cadmium reduction method and normalized to total cellular
protein.
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Cellular Attachment and Expression of the 67-Da LBP--
Having
established that the eNOS expression under shear stress is laminin
I-dependent, experiments were performed to prove whether
the 67-kDa LBP was involved.
Western blot experiments were done to show directly the expression of
the 67-kDa LBP. Fig. 5A shows
that regardless of the precoating, all cells were expressing the 67-kDa
LBP. Endothelial cells, exposed to shear stress on a laminin I matrix,
showed an increased expression of this LBP (see Fig.
5B).

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Fig. 5.
Western blots for the 67-kDa LBP.
Panel A, porcine aortic endothelial cells were grown under
static conditions either on pure glass or with a precoating of the
culture plate with collagen I, fibronectin, laminin I. Panel
B, porcine aortic endothelial cells grown on laminin I were
subjected to shear stress of 16 dyn/cm2 for 6 or 16 h.
Equal amounts of cell lysates were separated on reducing
SDS-polyacrylamide gel, blotted onto nitrocellulose, and probed with an
antibody specific for 67-kDa LBP.
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Attachment experiments were performed to evaluate the expression of a
functional LBP on the endothelial cell surface. For this reason, the
specific cellular binding to laminin I via 67 kDa was inhibited using
the peptide YIGSR, which represents a part of the short arm of the
1
chain of the laminin I molecule.
The cellular attachment to laminin I was reduced to approximately 50%
of the control by incubation with YIGSR (150 µg/ml) (see Fig.
6A), whereas the control
peptide YIGSK had no effect.

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Fig. 6.
Attachment experiments of endothelial cells
to laminin I. Panel A, simultaneous with the time point
of seeding the porcine aortic endothelial cells onto a laminin I-coated
plate the LBP inhibitory peptide YIGSR was added at a high
concentration (150 µg/ml). The subsequent cellular attachment was
recorded by counting the cells in 10 randomly distributed optical
fields. Each experiment was done in triplicate. Panel B,
shortly after first adhesion to laminin I (16 h) endothelial cells were
subjected to a shear stress of 16 dyn/cm2 for 6 h. The
addition of a high dose of the LBP inhibitory peptide (150 µg/ml)
resulted in an 80% loss of cells, whereas with no peptide addition
less than 10% of the cells was dislodged. Each experiment was done in
triplicate.
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This reduced attachment upon incubation with YIGSR was not a poisoning
effect of the peptide itself because proliferation assays of
endothelial cells supplemented with and without the peptide YIGSR
showed the same growth pattern (data not shown).
Similar results were obtained with the addition of high concentrations
of the peptide (150 µg/ml) 16 h after seeding and subsequent application of shear stress (16 dyn/cm2). After a 6-h shear
stress, 80% of the cells were dislodged in the presence of YIGSR,
whereas less than 10% of the cells were lost without the peptide (Fig.
6B).
Therefore, we used a reduced dose of only 35 µg/ml inhibitory peptide
YIGSR for the following Northern experiments. After 3 days of cell
culture, the time usually needed to reach confluent cell layers, the
endothelial cells had synthesized additional matrix proteins (see Fig.
1A), could withstand the shear stress, and less than 10% of
the cell population was washed off by the applied mechanical force
(data not shown).
To prove that the peptide YIGSR had no additional effect on endothelial
cells, proliferation experiments were performed. Endothelial cells that
were starved at 1% serum-containing medium for 16 h were
stimulated with 3 ng/ml basic fibroblast growth factor. There were no
differences between cells incubated with or without the peptide YIGSR,
indicating that viability as well as proliferation was not altered by
YIGSR (data not shown).
Inhibition of Shear Stress-induced eNOS Expression--
Both
semiquantitative reverse transcriptase-PCR and Northern analysis showed
that the original 2-fold laminin-dependent increase of the
eNOS expression after shear stress was abolished during incubation with
the peptide YIGSR (n = 5) (see Fig.
7). In contrast, control experiments with
the peptide YIGSK had no significant effect on the shear
stress-dependent expression pattern (2.3-fold induction,
n = 6).

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Fig. 7.
Influence of the YIGSR peptide on shear
stress-induced eNOS expression. RNA from porcine aortic
endothelial cells grown on laminin was inoculated without any peptides,
with the LBP-inhibitory peptide YIGSR, or with its control peptide
YIGSK. After an exposure of a shear stress of 16 dyn/cm2
for 6 h, total RNA was isolated, and 10-20 µg was separated on
a formaldehyde/formamide agarose gel. Videodensitometric quantification
of several Northern blots was analyzed as described. The densities of
specific eNOS mRNA bands of all experiments were related to their
corresponding GAPDH bands. The relative expression represents the eNOS
mRNA of the shear stress experiments normalized to their static
control, which was set at 1.
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These findings went along with experiments using the enzymatic laminin
fragments E8 or P1. In contrast to P1, the E8 fragment does not contain
the region where the 67-kDa laminin receptor binds to the molecule
because it represents only the "stem" portion of the laminin
cross-shaped trimer. Coating with the E8 fragment showed no induction
after a 6-h shear stress (1.2-fold over control, n = 2), whereas coating with P1 yielded a high response to the shear stress
(3.5-fold increase of expression, n = 3).
Effects of Incubations with YIGSR on eNOS Protein after Shear
Stress--
To verify the results of the previous mRNA analysis we
performed also Western blots against eNOS protein. Western blot
experiments for the different coatings collagen I, fibronectin, and
glass alone showed no induction of the eNOS protein after a 6-h shear stress (Fig. 3B shows representative bands of
n = 2). However, as in Fig. 3A, endothelial
cells grown on laminin I showed a remarked increase of eNOS
(n = 4). This protein induction was abolished completely by an incubation of the cells with the LBP-inhibitory peptide YIGSR during shear stress (n = 3) (see Fig.
8, A and B), whereas incubation with the control peptide YIGSK had no
effect (n = 3).

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Fig. 8.
Western blots for eNOS protein.
Panel A, representative Western blot of total cell lysates
from porcine aortic endothelial cells grown on laminin I-precoated
plates either under static control (C) conditions or under
16-dyn/cm2 shear stress with the LBP-inhibitory peptide
YIGSR (+R) or with the control peptide YIGSK (+K)
for 6 h. Panel B summarizes several experiments
(n = 3 for peptides and n = 4 without
peptides) that were analyzed densitometrically and normalized to their
corresponding static control.
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 |
DISCUSSION |
These experiments demonstrate that the application of shear stress
over several hours augments the expression of eNOS in porcine endothelial cells. The new finding in this study is that the
augmentation of eNOS expression depends on the presence of laminin I in
the extracellular matrix, suggesting a modulator role for it in the response of a vessel to mechanical forces. A 67-kDa LBP, which seems to
be constitutively expressed in the endothelium, acts as a functional
sensor at the basal side of the cell and is crucially involved in the
translation of the physical force to gene expression.
The increase of eNOS expression upon shear stress is in accordance with
earlier reports in cultured bovine aortic (3-6) and human umbilical
vein endothelial cells (5, 8, 30). Similar results have been reported
under in vivo conditions in lamb pulmonary arterial (9) and
rat aortic (10) as well as in canine coronary endothelial cells
(31).
Some groups observed an increase of eNOS expression in cells being
cultured on non-laminin matrices (3, 30). This is in apparent contrast
to our results because cultivating cells on fibronectin or pure glass
abolished the shear stress sensitivity in terms of eNOS expression.
This discrepancy may be explained by variations in the ability of the
cells to produce endogenous laminin. Our observation that cells taken
from the same pool produced high amounts of laminin I when grown on
plastic (irrespective of the coating) but not when cultivated on glass
plates, except when the glass plates were precoated by laminin,
supports the explanation above. Indeed, our Western blot data suggest a
laminin I-induced laminin production under these conditions.
Therefore, by cultivating endothelial cells on glass plates, a specific
role of laminin I could be detected when compared with other
matrix proteins. This is not an effect of laminin I alone because the
addition of laminin I to the superfusate did not change the eNOS
expression in control cells kept under static conditions (data not
shown). Only in combination with shear stress was there an effect (a
2-fold increase of eNOS expression). Interestingly, it has been
reported that endothelial cells under chronic shear stress produce more
laminin at the expense of fibronectin (21, 22). Additionally, laminin
expression correlates with endothelial cell differentiation during
angiogenesis (24, 32, 33). There is circumstantial evidence that eNOS
expression also correlates with endothelial cell
differentiation (34).
Binding of the cell to laminin I occurs not only via integrins,
especially those consisting of a
1- and one of several
-chains but also by a 67-kDa LBP (25-27). LBP was constitutively
expressed, regardless of the underlying matrix, which may reflect its
potential significance in mediating effects of matrix proteins on cell
signaling. The presence of a functioning LBP at the membrane level was
assessed indirectly by testing the adherence of freshly dispersed cells to a laminin matrix. High concentrations of the inhibitor of LBP binding, YIGSR (see below), reduced the adherence and led to an extensive detachment of freshly attached cells under shear stress. This
may be the result of the lack of other attachment substrates. After
2-3 days in culture and using much less peptide, this shear-induced detachment was not observed, indicating that binding sites other than
LBP are more important for permanent attachment and firm cell
adherence. These hypothesis is supported by macroscopic inspection of
the matrix protein compositions. The Coomassie-stained protein gels
showed no obvious differences among the various pretreatments of the
culture plates. However, when probed in Western blots there was a clear
distinction between the laminin coating and collagen and fibronectin
coating. This suggests that the role of LBP is not primarily in cell
adhesion but perhaps in sensing external signals applied to the cell.
Even after several days in culture, the laminin I-dependent
eNOS expression could still be inhibited by the addition of YIGSR, a
peptide deduced from the LBP binding site (27) on the laminin I
molecule. When applied in high concentrations, this peptide was shown
to attenuate tumor invasion and vasculogenesis (35) in vivo,
therefore suggesting a role for this protein in development and under
pathophysiologic conditions. In the present experiments, a much smaller
concentration of this peptide inhibited the eNOS induction on
transcriptional and translational level completely. This inhibition was
highly specific because the nearly identical control peptide had no
effect. The central role of LBP in mediating the shear stress response
is supported further by preliminary experiments using the laminin
fragments P1 and E8 for coating the glass plates. Like intact laminin,
the P1 fragment also resulted in a shear stress-induced increase of
eNOS expression. Fragment P1 represents the "upper" part of the
cross-shaped laminin molecule containing the YIGSR binding site (36).
In contrast, coating with the fragment E8 of laminin, which represents
the large stem of the protein that does not contain the binding site
for LBP (36), led to a loss of the shear stress sensitivity of the
cells in terms of eNOS expression.
The mechanism by which LBP translates shear stress into gene
expression is not yet clear. Massia et al. (37) showed the colocalization of the 67-kDa LBP with stress fibers of the cytoskeleton and, furthermore, with part of the focal adhesion kinase complex,
-actinin and vinculin. This colocalization suggests that LBP is
involved in signaling processes located in focal adhesion points. In
this context, it should be emphasized that shear stress induces a very
dynamic change of focal adhesion points soon after initiation of shear
stress (14). Our results do not exclude that additional membrane
structures are involved in the signal transduction process. This could
explain why there was a small but significant increase of eNOS
expression also on a collagen I matrix, although we did not find a
corresponding translation to eNOS protein. Recently, Ardini et
al. (38), as well as Halatsch et al. (39), described a
close coregulation and physical association of the 67-kDa LBP and the
integrin
6
4 in the human carcinoma cell
line A43. This very close association of the different receptors,
possibly created by binding to the same laminin molecule, could be a
crucial step in the translation of the signal which is currently being
studied. Even though some steps of the signal cascade are still
missing, it is reasonable to assume that the increase of eNOS
expression was caused by enhanced transcription rather than a
modification of the half-life time of the eNOS mRNA. The shear
stress-induced increase of mRNA expression was abolished after
incubation with actinomycin D, which inhibits mRNA production. In
fact, the increase in eNOS expression went along with a substantial
increase of eNOS protein synthesis as revealed by Western blotting.
Measurements of elevated NOX concentration in
the conditioned media confirmed that this eNOS protein was
physiologically functional.
In conclusion, this study demonstrated that a cell-laminin I
interaction via a 67-kDa LBP is an important step in the signal transduction pathway by which shear stress elicits eNOS expression. This function suggests a physiological role for a protein that, up to
this point, was considered important mainly for pathophysiologic processes such as tumor invasion.