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1
* Houston VA Medical Center and the Departments of Medicine; and
Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA
1Correspondence: Houston VA Medical Center, Bldg. 109, Room 130, 2002 Holcombe Blvd., Houston TX 77030, USA. E-mail:wdurante{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: biomechanical strain • L-proline • cationic amino acid transporter • arginase • vascular cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cellular L-arginine requirements are met primarily by
the uptake of extracellular L-arginine via specific membrane
transporters. In vascular smooth muscle cells (SMC) the uptake of
L-arginine is mediated by the system y+ carrier
(2
, 3)
. This Na+-independent
transport system possesses high affinity for cationic amino acids and
is stimulated by the presence of substrate on the opposite
(trans) side of the membrane (4)
. Recently, the
proteins responsible for the activity of the system
y+ carrier have been identified and designated as
cationic amino acid transporter-1 (CAT-1), CAT-2, CAT-2A, and CAT-3
(5
6
7
8
9)
. CAT-1, CAT-2, and CAT-3 are kinetically
indistinguishable transporters that have a high affinity
(Km
100 µM) for L-arginine (5
6
7
8)
.
In contrast, CAT-2A is a kinetically distinct alternate splice variant
of CAT-2 that possesses low affinity (Km 1–2 mM)
but high transport capacity for L-arginine (9)
. Previous
studies in our laboratory and others have demonstrated that SMC express
only CAT-1 and CAT-2 mRNA and that specific biochemical stimuli can
induce the expression of these two transporters (10
11
12
13
14)
.
L-arginine is metabolized via two distinct pathways in vascular SMC.
The inducible NO synthase (iNOS) enzyme converts L-arginine to the
diatomic signaling gas NO. This enzyme is induced after stimulation
with inflammatory cytokines and results in the generation of cytotoxic
levels of NO (15
16
17)
. Alternatively, L-arginine is
catabolized to L-ornithine and urea by the enzyme arginase
(18
19
20)
. At least two distinct isoforms of mammalian
arginase have been identified. Type I arginase is a cytosolic enzyme
that is highly expressed in the liver as well as other tissues and
constitutes a majority of total arginase activity (21
, 22)
. In contrast, type II arginase is a mitochondrial enzyme
that is expressed at lower levels in several extrahepatic tissues
(22)
. Finally, the arginase metabolite L-ornithine is
metabolized to important regulatory molecules. Ornithine decarboxylase
(ODC) converts L-ornithine to the polyamine putrescine, which plays a
critical role in the mitogenic response of vascular SMC (10
, 23
, 24)
. In addition, L-ornithine is catabolized by the
mitochondrial enzyme ornithine aminotransferase (OAT) to
pyrroline-5-carboxylate, which is further metabolized to L-proline that
is essential for the synthesis of many structural proteins, including
collagen (25)
.
Vascular SMC are continuously exposed in vivo to cyclic
stretch, which arises from the periodic change in vessel diameter as a
result of pulsatile blood flow. Recent studies indicate that cyclic
stretch (or strain) exerts significant effects on SMC function. The
imposition of cyclical mechanical stretch alters SMC orientation and
may play a fundamental role in governing SMC phenotype (26
, 27)
. In addition, cyclic stretch may influence arterial
remodeling by regulating SMC proliferation and extracellular matrix
production (28
29
30
31
32
33
34)
. Since L-arginine can be metabolized
to important growth stimulatory polyamines and to the essential matrix
component L-proline, the present study examined the effect of cyclic
stretch on L-arginine transport and metabolism by vascular SMC. We now
report that a physiologically relevant level of cyclic stretch
stimulates the transport of L-arginine and specifically directs its
metabolism to the integral collagen constituent L-proline and away from
the formation of polyamines. Cyclic stretch mediates this effect by
inducing CAT-2 and arginase I gene expression while simultaneously
blocking iNOS and ODC activity. In addition, we demonstrate that the
cyclic stretch-mediated increase in type I collagen formation is
dependent on L-proline synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]UTP (400 Ci/mmol) were from Amersham
Life Sciences (Arlington Heights, Ill.).
SMC culture
Vascular SMC were isolated by elastase and collagenase digestion
of rat thoracic aorta and characterized by immunological and
morphological criteria, as described previously (35)
.
Cells were cultured serially in minimum essential medium containing
Earle’s salts, 5.6 mM glucose, 2 mM L-glutamine, 20 mM TES-NaOH, 20 mM
HEPES-NaOH, and 100 units/ml of penicillin, streptomycin, and neomycin.
Subcultured strains were used between passages 4 and 29.
Cyclic stretch
SMC were plated onto six-well, flexible-bottomed plates coated
with type I collagen (Flexercell Corp. McKeesport, Pa.). The thickness
of the silicon elastomer on the bottom of the plates varies along the
diameter of the wells such that a near-homogeneous strain profile is
obtained throughout the membrane. When cells reached confluence, the
culture media were replaced with serum-free media containing BSA
(0.1%) for 24 h. Cells were then subjected to mechanical
deformation with the Flexercell Strain Unit (FX 3000, Flexercell
Corp.). This stress unit is a modification of the unit initially
described by Banes et al. (36)
and consists of a
computer-controlled vacuum unit and a baseplate to hold the culture
dishes. Vacuum is repetitively applied to the bottom of the dishes via
the baseplate, which is placed in a humidified incubator with 5%
CO2 at 37°C. The computer system controls the
frequency of deformation and the negative pressure applied to the
culture plates. SMC were exposed to an equiaxial stretch of 10% at a
frequency of 1 Hz.
L-arginine transport
L-arginine transport was determined by measuring the influx of
radiolabeled L-arginine into SMC (3)
. Cells were washed
with HEPES buffer (140 mM choline chloride, 5.6 mM D-glucose, 5.0 mM
KCl, 1.0 mM MgCl2, 0.9 mM
CaCl2, and 25 mM HEPES, pH 7.4) and incubated for
45 s in HEPES buffer containing
[3H]L-arginine (50 µM, 1 µCi). Transport
activity was terminated by aspirating the media and washing the cells
in ice-cold HEPES buffer. Cell-associated radioactivity was extracted
with 0.2% SDS in 0.2 N NaOH and then assayed by liquid scintillation
spectrometry. To correct for nonspecific uptake, cells were incubated
in parallel with HEPES buffer containing 10 mM unlabeled L-arginine and
the fraction of the radioactivity of the cells was determined; this
fraction was then subtracted from each data point.
Arginase activity
Arginase activity was determined by monitoring the formation of
[14C]urea from
[guanido-14C]L-arginine
(13)
. SMC were scraped in ice-cold phosphate-buffered
saline (PBS), centrifuged at 1000 g for 5 min at 4°C, and
sonicated in Tris buffer (10 mM, pH 7.4) containing Triton X-100
(0.4%), leupeptin (10 mg/ml), and aprotinin (10 mg/ml). An aliquot of
the cell lysate (100 µg protein) was then added to an equal volume of
Tris buffer (10 mM, pH 7.4) containing MnCl2 (10
mM) and arginase was activated by heating for 10 min at 56°C. The
arginase reaction was initiated by adding Tris buffer (10 mM, pH 9.6)
containing L-arginine (10 mM) and
[guanido-14C]L-arginine (0.25 Ci),
and samples were incubated at 37°C for 30 min. Reactions were
terminated by adding ice-cold sodium acetate (250 mM, pH 4.5)
containing urea (100 mM). [14C]Urea was
separated from basic amino acids by Dowex chromatography and
[14C]urea formation was quantified by
scintillation counting.
iNOS expression
The expression of iNOS by SMC was assessed by monitoring iNOS
protein expression and measuring iNOS activity. iNOS protein levels
were determined by Western blotting using anti-mouse iNOS IgG, as
described previously (37)
. For iNOS activity, SMC were
collected in Tris buffer (50 mM, pH 7.4) containing EDTA (0.5 mM), EGTA
(0.5 mM), DDT (1 mM), tetrahydrobiopterin (1 µM), leupeptin (10
mg/ml), and PMSF (50 µg/ml). Cells were lysed by sonication and the
homogenates were centrifuged at 14,000 g for 15 min at
4°C. The supernatant (50 µg protein) was incubated in HEPES buffer
(50 mM, pH 7.4) containing L-arginine (100 µM),
[3H]L-arginine (1 µCi),
CaCl2 (1.25 mM), NADPH (0.5 mM), EDTA (1 mM),
flavin adenine dinucleotide (10 mM), flavin mononucleotide (5 µM),
and tetrahydrobiopterin (10 µM) for 30 min at 37°C. Incubations
were terminated by the addition of ice-cold HEPES buffer (20 mM, pH
5.5) and EDTA (5 mM). [3H]L-citrulline was
separated from [3H]L-arginine by Dowex-50W X8
chromatography and radioactivity was quantified by scintillation
counting. iNOS activity was expressed as a percentage of the maximum
response obtained in each experiment.
ODC assay
SMC were harvested in ice-cold Tris buffer (20 mM Tris, 0.1 mM
EDTA, 2 mM DDT, and 0.1 mM pyridoxal-5-phosphate, pH 7.4), sonicated,
and centrifuged at 14,000 g for 20 min at 4°C. The
supernatant was collected and ODC activity was determined by measuring
the release of 14CO2 from
[1-14C]L-ornithine, as described previously
(13)
.
OAT assay
SMC lysates were prepared by sonication in Tris buffer (50 mM
Tris, 0.2% Triton X-100, 50 µM pyridoxal-5-phosphate, 10 mg/ml
leupeptin, and 50 µM PMSF, pH 8.0) and OAT activity was determined by
measuring the formation of pyrroline-5-carboxylate. Standard assay
mixtures (1 ml) contained 35 mM L-ornithine, 5 mM -ketoglutarate, 50
µM pyridoxal-5-phosphate, and cell lysate (100 µg) in Tris buffer.
After incubation at 37°C for 20 min, the reaction was stopped by
adding 3 N perchloric acid and 2% ninhydrin. After heating for 5 min
in a boiling water bath, a reddish pigment was precipitated by
centrifugation and dissolved in ethanol. Pyrroline-5-carboxylate
production was determined by absorbance spectrophotometry at 510 nm
using an extinction coefficient of 16.2 mM-1 ·
cm-1 (38)
.
L-proline and polyamine production
L-proline and polyamine formation were determined by incubating
SMC with [3H]L-arginine and measuring the
intracellular formation of radiolabeled L-proline and putrescine,
respectively (13
, 14)
. SMC were incubated with
[3H]L-arginine(20 µCi/mmol) and the reactions
were stopped at various times by removing the media and washing the
cells with ice-cold PBS. Cells were solubilized in Tris buffer (20 mM,
pH 7.4) containing Triton X-100 (0.01%), spotted onto thin-layer
chromatography plates, and developed in the solvent system
chloroform:methanol:ammonium hydroxide:water (1:4:2:1, by volume).
L-proline and putrescine were detected by ninhydrin spray and the
[3H]L-arginine metabolites were identified with
cochromatography with unlabeled standards. Plates were then scraped,
and L-proline and putrescine generation was determined by liquid
scintillation counting.
Generation of arginase probes
Arginase cDNA fragments were amplified from SMC by reverse
transcriptase-polymerase chain reaction (RT-PCR) (10)
.
Primers were designed according to the published sequence of rat
arginase I and arginase II (18
, 19)
. The forward
5'-TAGAGAAAGGTCCCGCAGCAT-3' and reverse 5'-TGCTTCCAATTGCCATACTGTG-3'
primers were used to amplify a 252-bp arginase I transcript while the
forward 5'-CCTAGTGAAGCTGCGAACGTG-3' and reverse
5'AGAGAAAGGGGCTCCGACTACA-3' primers were used to amplify a 197-bp
arginase II transcript. The cDNA was amplified in a reaction mixture
containing MgCl2, dATP (0.2 mM), dTTP (0.2 mM),
dGTP (0.2 mM), dCTP (0.2 mM), arginase primers (50 pmol each),
Taq DNA polymerase (2.5 U/ml) in standard reaction buffer.
Amplification consisted of 30 cycles of PCR [1 min at 94°C for
denaturing, 40 s at 60°C for annealing, and 90 s and 5 min
(final cycle) at 75°C for elongation. Products of PCR amplification
were resolved by agarose gel electrophoresis, stained with ethidium
bromide, visualized on an UV transilluminator, and photographed.
Products of expected size were subcloned into pCRII plasmids
(Invitrogen, San Diego, Calif.) and sequenced to confirm their identity
and orientation.
mRNA analysis
mRNA levels were determined by ribonuclease protection assay
using a commercially available kit (Ambion Inc.). Total RNA (15 µg)
was hybridized with 
1 x 106 cpm of
[32P]UTP-labeled antisense CAT, arginase, and
GAPDH (316-bp) riboprobes. The CAT-1 (195-bp) and CAT-2 (210 bp)
antisense RNA probes were prepared as described earlier (10
, 13)
. Protected RNA was analyzed by electrophoresis using 6%
acrylamide/8 mM urea gels. Gels were exposed overnight to X-ray film at
-70°C in the presence of intensifying screens. The size of the
protected nucleotide fragments was confirmed using a
[32P]-labeled RNA ladder. Relative mRNA levels
were quantified by scanning densitometry (LKB 2222–020 Ultrascan
laser densitometer, Bromma, Sweden) and normalized with respect to
GAPDH mRNA.
Collagen synthesis
Collagen synthesis was determined by the collagenase-digestible
method of Peterkofsky and Diegelman (39)
. SMC were pulsed
with [2,3,4,5-3H]L-proline (2 µCi/ml) in the
presence of ascorbic acid (50 µg/ml) and ß-aminopropiononitrile (80
µg/ml) for 24 h. Culture media were then removed and added to an
equal volume of cold Tris buffer (0.1 mM, pH 7.4) containing 0.65 mM
NaCl, 5.0 mM CaCl2, 2.5 mM N-ethylmaleimide, and
100 µg/ml BSA. Trichloroacetic acid (10%) was then added to an
aliquot of the sample and the material was allowed to flocculate for 30
min at 4°C. The sample was then centrifuged at 14,000 g
for 10 min at 4°C and the pellet was washed twice with cold 95%
ethanol, dried, dissolved in 0.2% SDS in 0.2 N NaOH, and assayed by
liquid scintillation counting. A second aliquot was digested with
highly specific collagenase form III (10 U/ml) for 90 min at 37°C and
then treated identically as the nondigested aliquot.
Type I collagen formation
For type I collagen determination, culture media were collected,
concentrated using centricon YM30 filters (Amicon Inc., Beverly,
Mass.), and proteins were solubilized with electrophoresis buffer [125
mM Tris-HCl (pH 6.8), 2% SDS, 12.5% glycerol, 1%
ß-mercaptoethanol, and trace bromphenol blue]. Samples were boiled
for 10 min and proteins were separated by SDS-polyacrylamide gel
electrophoresis using 6% gels. Gels were then electrophoretically
transferred to nitrocellulose membranes, blocked for 1 h at room
temperature in Tris buffer (50 mM, pH 7.4) containing 1% BSA, and
incubated with rabbit anti-collagen type I antibody (1 µg/ml) for
1 h. Membranes were then washed in Tris buffer, incubated for
1 h at room temperature with anti-rabbit horseradish peroxidase
conjugated antibody (1:5,000 dilution), and incubated with
chemoluminescence reagents. Type I collagen levels were quantified by
scanning densitometry.
Statistics
Results are expressed as the means ± SE.
Statistical analysis was performed with the use of a Student’s
two-tailed t test and an analysis of variance when more than
two treatments were compared. Values of P<0.05 were
considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A significant increase in transport activity (compared to
nonstretched static SMC) was observed after 24 h of mechanical
stretch and was maintained during 72 h of cyclic stretch.
Figure 2
shows a representative Eadie-Hofstee plot demonstrating that saturable,
high-affinity uptake of L-arginine by vascular SMC was mediated by a
single carrier. Data from several experiments (n=5)
indicated that this transporter had a Michaelis constant
(Km) of 41.2 ± 5.2 µM and a maximum
transport velocity (Vmax) of 545 ± 48 pmol/mg protein/45 s. Imposition of cyclic stretch for 72 h
significantly (P<0.05) increased both the
Km (72.4±8.8 µM) and
Vmax (905±102 pmol/mg protein/45 s)
of L-arginine transport by vascular SMC.
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Ribonuclease protection assays demonstrated low levels of CAT mRNA
expression in control static SMC (Fig. 3
). However, application of cyclic stretch markedly elevated CAT-2 mRNA
levels. A peak sixfold increase in CAT-2 mRNA was observed after 2 h of cyclic stretch and CAT-2 message remained elevated throughout the
72 h of mechanical strain (Fig. 3)
. In contrast, cyclic stretch
had no effect on the expression of CAT-1 mRNA (Fig. 3)
.
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Exposure of vascular SMC to cyclic stretch also modulated L-arginine
metabolism. A significant increase in arginase activity was detected
within 2 h of cyclic stretch and was maintained during 72 h
of cyclic stretch (Fig. 4
). Ribonuclease protection analysis revealed that cyclic strain induced
an increase in arginase I mRNA levels that peaked after 6 h
(30-fold) of cyclic stretch, then decaying to basal levels by 48 h
(Fig. 5
). Ribonuclease protection assays failed to detect arginase II mRNA in
either static or stretched cells (data not shown).
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Incubating SMC with interleukin-1ß for 24 h induced the
expression of iNOS protein and activity in a concentration-dependent
manner (Fig. 6
). However, the application of cyclic stretch markedly reduced the
cytokine-mediated induction of iNOS protein and activity (Fig. 6)
. In
the absence of interleukin-1ß, cyclic strain had no effect on iNOS
protein or activity (Fig. 6)
.
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In addition to stimulating L-arginine transport and L-arginine
metabolism to L-ornithine, cyclic stretch exerted differential effects
on L-ornithine metabolism. The application of cyclic stretch for up to
48 h had no effect on ODC or OAT activity (data not shown).
However, 72 h of cyclic stretch significantly decreased ODC
activity but had no effect on OAT activity (Fig. 7
). Cyclic stretch also increased the capacity of SMC to generate
L-proline from extracellular L-arginine in a time-dependent manner
(Fig. 8A
). The latter experiments were based on the rationale that
[3H]L-arginine must be converted to
[3H]L-ornithine by arginase and then
metabolized to [3H]L-proline and
[3H]putrescine by the enzymes OAT and ODC,
respectively. The stimulatory effect of cyclic stretch on L-proline
synthesis was inhibited by the cationic amino acid transport inhibitor
L-NMA (10 mM) (3)
, by the arginase inhibitor L-NOHA (1 mM)
(40)
, and by the specific OAT inhibitor L-canaline (100
µM) (41)
(Fig. 8B
). In contrast, cyclic
stretch inhibited the production of polyamines from L-arginine in a
time-dependent manner (Fig. 8C
).
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Finally, application of cyclic stretch to SMC stimulated collagen
synthesis (Fig. 9
) and the secretion of type I collagen by approximately twofold
(Fig. 10
). The addition of L-canaline (100 µM) to SMC abolished the cyclic
stretch-induced production of type I collagen without affecting the
synthesis of collagen from static control cells (Fig. 10)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In particular, cyclic stretch increases the transcellular transport
of L-arginine and the intracellular metabolism of L-arginine to
L-ornithine by stimulating the expression of the genes for CAT-2 and
arginase I while inhibiting the expression of the iNOS gene.
Furthermore, cyclic stretch stimulates the intracellular metabolism of
L-ornithine to L-proline and away from polyamines by inhibiting ODC
activity. Moreover, cyclic stretch-mediated production of collagen by
vascular SMC is dependent on L-proline synthesis. Thus, cyclic stretch
coordinately promotes the availability of intracellular L-arginine
substrate and selectively diverts its metabolism to collagen synthesis.
|
Cyclic stretch stimulates the transport of L-arginine in a
time-dependent manner. Kinetic experiments indicate that high affinity
L-arginine (Km50 µM) transport is mediated by a
single carrier and that mechanical forces increase both the
Vmax and Km of this
transporter. These kinetic data suggest that the increase in transport
activity likely arises from the de novo synthesis of
additional transport proteins. Consistent with this, we found that
cyclic stretch induced CAT-2 mRNA expression. The selective induction
of CAT-2 by cyclic stretch is also observed after the administration of
inflammatory cytokines (12
, 42)
and may provide an
important adaptive mechanism in elevating the intracellular levels of
L-arginine during physiological conditions of biomechanical or
biochemical stress.
In addition to stimulating transcellular L-arginine transport, cyclic
stretch stimulates the intracellular metabolism of L-arginine to
L-ornithine in vascular SMC. In this study we have found that
mechanical strain induces an increase in arginase activity, which is
accompanied by a selective increase in arginase I mRNA, suggesting that
arginase I mediates the stretch effect. In contrast, cyclic stretch
attenuates cytokine-stimulated SMC NO synthesis by inhibiting iNOS
protein expression. This latter finding complements a recent study
showing that cyclic stretch suppresses iNOS expression in cardiac
myocytes (43)
. However, it contrasts with an earlier study
demonstrating that cyclic stretch stimulates the transcriptional
activation of the endothelial NOS gene (44)
. Thus,
biomechanical strain exerts divergent isoform-specific regulatory
effects on the expression of NOS. The ability of cyclic stretch to
down-regulate the expression of iNOS may play an important
cytoprotective role since high-output NO synthesis by the iNOS enzyme
has been shown to induce SMC apoptosis (16
, 17)
. In
addition, the coordinate stimulation of L-arginine transport and
inhibition of iNOS expression may provide a mechanism by which
increased levels of substrate (L-arginine) are provided to SMC during
stretch-induced activation of arginase.
We reasoned that the capacity of cyclic stretch to stimulate arginase
activity may function to direct L-arginine to L-ornithine metabolism to
generate biologically relevant L-proline. In support of this
hypothesis, we found that exposure of SMC to cyclic stretch resulted in
a prominent increase in the capacity of SMC to generate L-proline from
extracellular L-arginine. This stretch-mediated effect is blocked by
the cationic amino acid transport inhibitor L-NMA, and the arginase
inhibitor L-NOHA, indicating that both the transcellular transport of
L-arginine and intracellular arginase activity are limiting factors
that govern the ability of cyclic stretch to generate L-proline. In
addition, the formation of L-proline was blocked by the OAT inhibitor
L-canaline, demonstrating that OAT activity is also required for the
generation of L-proline. Cyclic stretch does not directly stimulate OAT
activity. However, given the low affinity (3 mM) (45)
of OAT for L-ornithine, it is likely that cellular L-proline production
is substrate-limited. Thus, by increasing intracellular L-ornithine
synthesis and by blocking ODC activity, cyclic stretch may provide
additional substrate for synthesis of L-proline. In support of this
notion, the highest levels of L-proline synthesis are observed after
72 h of cyclic stretch, at which time ODC activity is inhibited.
The level of cyclic stretch (10% at 1 Hz) required to stimulate
L-arginine transport and metabolism in our study may be physiologically
relevant. During the cardiac cycle, the maximum stretch of the human
aorta has been reported to be between 9 and 12% under normotensive
conditions (46)
. Similar levels of cyclic stretch are
observed in other large peripheral vessels, including brachial,
femoral, and pulmonary arteries (47)
. However,
extrapolation of these in vitro cell monolayer experiments
to the more complex 3-dimensional blood vessel requires caution.
Consistent with earlier studies (32
, 33
, 34)
, we
demonstrated that cyclic stretch stimulates collagen synthesis and the
release of type I collagen from vascular SMC. However, we also found
that the OAT inhibitor L-canaline blocks cyclic stretch-mediated
collagen production, suggesting that endogenous L-proline synthesis is
required for collagen synthesis by cyclic strain. In addition, we found
that cyclic stretch inhibits the synthesis of NO, an inhibitor of
collagen formation (48)
. Thus, by differentially
regulating the synthesis of L-arginine metabolites, cyclic stretch may
play a critical role in promoting collagen synthesis. Moreover, these
cyclic stretch actions that promote collagen accumulation are further
amplified by the cyclic stretch-mediated suppression of the type I
collagen-degrading enzyme metalloproteinase-1 (49)
. The
ability of cyclic stretch to stimulate collagen synthesis and
simultaneously inhibit matrix metalloproteinase expression may play an
important role in stabilizing vascular lesions and preventing plaque
rupture (50)
.
Polyamines play an integral role in the mitogenic response of vascular
SMC. Both arterial injury-induced and growth factor-mediated SMC
proliferation are associated with striking increases in ODC activity
and polyamine formation (10
, 14
, 24)
. Moreover, inhibition
of ODC activity blocks SMC proliferation both in vitro and
in vivo (10
, 14
, 51)
. The present study found
that prolonged cyclic stretch (72 h) blocks ODC activity and polyamine
synthesis, suggesting a possible role for mechanical strain in
regulating SMC growth. Although studies examining the effect of cyclic
stretch on SMC growth have yielded contradictory results
(28
29
30
31)
, we recently found that application of 10%
cyclic stretch to vascular SMC markedly attenuates their proliferative
response (31)
. Thus, the capacity of cyclic stretch to
regulate L-arginine metabolism may also play a pivotal role in
maintaining vascular SMC in a quiescent nonproliferative state.
In conclusion, these studies demonstrate that a physiologically
relevant level of cyclic stretch stimulates the transport and
metabolism of L-arginine by stimulating the genes for CAT-2 and
arginase I, respectively, while inhibiting the gene for iNOS. In
addition, cyclic stretch directs the metabolism of L-ornithine from the
formation of growth stimulatory polyamines to the essential collagen
component L-proline by blocking ODC activity. As shown in the model in
Fig. 11
, these multiple sites of action of cyclic stretch appear to be
coordinately directed toward stimulating collagen synthesis and
inhibiting SMC proliferation. The ability of cyclic stretch to regulate
L-arginine transport and metabolism may serve to promote vascular
homeostasis in both normal arteries and in arterial disease by
stabilizing vascular lesions and limiting SMC proliferation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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stimulate the cat-2 gene of the L-arginine transporter in cultured vascular smooth muscle cells. J. Biol. Chem. 271,11280-11283 in vascular endothelium. Proc. Assoc. Am. Phys. 108,356-361[Medline]
-transaminase. J. Biol. Chem. 240,1225-1230This article has been cited by other articles:
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  J.-M. Tadie, P. Henno, I. Leroy, C. Danel, E. Naline, C. Faisy, M. Riquet, M. Levy, D. Israel-Biet, and C. Delclaux Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L489 - L497. [Abstract] [Full Text] [PDF]
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 X. Jiang, F. Yang, E. Brailoiu, H. Jakubowski, N. J. Dun, A. I. Schafer, X. Yang, W. Durante, and H. Wang Differential Regulation of Homocysteine Transport in Vascular Endothelial and Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1976 - 1983. [Abstract] [Full Text] [PDF]
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) and in SMC
subjected to cyclic stretch for 72 h (•). Similar findings were
made in 5 separate experiments.
)
or presence (+ or 
) of cyclic stretch. The iNOS immunoblot is
representative of three separate experiments; iNOS activity is
expressed as the means ± SE of 4 separate
experiments. *Statistically significant effect of cyclic stretch.
