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INTRODUCTION |
The vascular smooth muscle cell plays a prominent role in
development and maintenance of arterial structure. Vascular smooth muscle cells are the primary source of arterial extracellular matrix
(ECM),1 including collagens,
elastic fibers, and several proteoglycans (1). Proteoglycans serve
several functions in the artery wall, including regulation of cell
adhesion, migration, and proliferation (2-4). The major proteoglycan
in the arterial ECM synthesized by arterial smooth muscle cells (ASMC)
is the large chondroitin sulfate proteoglycan versican, also known as
PG-M (5-9). Versican is a member of a family of proteoglycans
including brevican, neurocan, and aggrecan that can bind hyaluronan and
form large aggregates (10, 11). These large aggregates contribute to
tissue mechanical properties, providing a hydrated sponge-like matrix
that resists or cushions against deformation (12). Arteries also
contain smaller proteoglycans that contain dermatan sulfate
glycosaminoglycans such as decorin and biglycan, which interact with
other ECM proteins and with macromolecules that enter the vascular wall
such as low density lipoproteins (13). In addition, blood vessels
contain perlecan, which is a heparan sulfate proteoglycan associated
with basal lamina surrounding ASMC (2).
Vascular smooth muscle cells are under dynamic mechanical stresses from
arterial pressure, and their responses to mechanical stimuli have
therefore been of long-standing interest. In the past decade,
improvements in bioengineering have provided much more
precise and uniform methods of cell deformation (14). Using DNA
microarrays and a device that provides a precise and uniform biaxial
strain profile, we have shown that small mechanical deformations, well
below the amplitudes that cause cell injury, induce highly specific
molecular events in ASMC (15). These events include induction of
several genes that may affect arterial extracellular matrix, including
tenascin-C and plaminogen activator inhibitor-1. In addition, small
deformations specifically suppress matrix metalloproteinase-1, an
enzyme that can initiate degradation of fibrillar collagen (16).
These studies indicate that deformation regulates smooth muscle cell
ECM metabolism and suggest that ASMCs may modify their biomechanical
environment in a manner that limits potential biomechanical injury.
Because proteoglycans are ECM molecules that can play a prominent role
in tissue mechanics, this study was designed to determine whether
mechanical strain induced specific changes in the synthesis of
proteoglycans and altered their ability to interact with other ECM molecules.
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EXPERIMENTAL PROCEDURES |
Materials and Cell Culture--
ASMC were prepared from explants
from excess aortic tissue from the donor at the time of organ harvest
for orthotopic cardiac transplantation at Brigham and Women's
Hospital. ASMC were maintained in Dulbecco's modified essential
medium, 10% fetal calf serum and 1% penicillin/streptomycin sulfate
(16) at 37 °C, 5% CO2 up to passage 6-7 for
experiments. The Brigham and Women's Hospital Committee for Human
Research approved the protocol.
Mechanical Strain--
Mechanical deformation was applied to a
thin and transparent membrane on which cells were cultured, an approach
that provides a nearly homogeneous biaxial strain profile. Each culture
dish consists of a plastic cylinder and a circular silicone
elastometric membrane, which is the culture surface. The membrane
undergoes cyclic tensile deformation as the platen assembly moves
sinusoidally. We have previously measured membrane strains with a
high-resolution video device (17); for this study, all experiments were
performed with 4% cyclic strain, a magnitude of strain that does not
lead to cell injury but reproducibly induces a restricted set of genes (15).
Culture membranes were precoated with 2 µg/ml serum fibronectin in 13 ml of Hank's solution for 24 h at 4 °C and then washed twice
with 10 ml of phosphate-buffered saline. ASMC were plated on the coated
membrane dish at a density of 6 × 105 cells/dish in
13 ml of Dulbecco's modified essential medium containing 10% fetal
bovine serum and incubated for 24 h. Before mechanical strain was
applied, 10 ml of fresh medium was exchanged. To eliminate the variable
of time-dependent changes because of cell age or effects of
adhesion to fibronectin or the membrane in each experiment, all cells
were cultured on the membrane for an identical time period, and cells
and media from all samples were harvested at the same time. For
example, in a time course experiment with strain, the time point
represents the time prior to harvest that strain was initiated, such
that the strain sample and control sample were harvested at the same time.
Metabolic Labeling and Proteoglycan Isolation--
To assess
proteoglycan synthesis, cells were labeled with 100 µCi per ml
Na2[35S]O4 for various times. The
medium was combined with a 0.1 volume of 10× protease inhibitors
dissolved in 8 M urea buffer (8 M urea, 2 mM EDTA, 0.25 M NaCl, 50 mM
Tris-HCl, and 2% Triton X-100 detergent, pH 7.4, Ref. 8). The cell
layer was washed with phosphate-buffered saline and scraped into 8 M urea buffer with 1× protease inhibitors (5 mM benzamidine, 10 mM 6-aminohexanoic acid, and
1 mM phenylmethylsulfonyl fluoride, Ref. 18). Total
[35S]sulfate incorporation into proteoglycans was
determined by CPC precipitation (19). Medium and cell layer extracts
were purified and concentrated by ion exchange chromatography on
DEAE-Sephacel in 8 M urea buffer and eluted with 8 M urea buffer containing 3 M NaCl (20).
Enzymatic Digestion and Electrophoresis--
Aliquots of
DEAE-purified material containing 30,000 dpm 35S were
precipitated in 80% ethanol and 1.3% potassium acetate. The resulting
pellet was then applied directly to SDS-PAGE or digested by incubation
prior to chromatography with 2.3 units per ml chondroitin ABC lyase
(Sigma) in Tris-buffered solution (45 mM Tris, 0.09 mg/ml
bovine serum albumin, 2.7 mM sodium acetate, pH 8.0, Ref. 21), or in heparitinase I and II (Sigma) in Tris-buffered solution (45 mM Tris, 0.09 mg/ml bovine serum albumin, 10 mM
calcium acetate, pH 7, Ref. 22).
Column Chromatography--
To determine the size classes of
[35S]sulfate-labeled proteoglycans synthesized and
secreted by the cells, medium and cell layer extracts, purified and
concentrated over DEAE-Sephacel, were applied to 8 mm × 113 cm-Sepharose CL2B molecular sieve column in 4 M guanidine
buffer (4 M guanidine, 10 mM EDTA, 0.5% Triton
X-100 detergent, 50 mM sodium acetate, pH 7.4) and
collected in 0.5-ml fractions (23).
Northern Analysis--
Total RNA was isolated using guanidine
isothiocyanate solubilization followed by phenol extraction at pH 4 and
subsequent precipitation in isopropyl alcohol (24). Purified samples
were fractionated on 1.0% formaldehyde-agarose gels, alkali denatured in 50 mM NaOH, 10 mM NaCl, and transferred to
nylon blotting membranes (Zeta Probe; Bio-Rad Laboratories, Richmond,
CA). Blots were hybridized with cDNA probes to the following matrix
molecules: human versican, clone 7 (25); human biglycan (26), clone
p16; human collagen type I, clone HF677 (27); human perlecan, clone
HS-1 (28), and bovine decorin (29). These were generous gifts from
Erkki Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA; Larry Fisher, National Institute of Dental Research, Bethesda, MD;
Francisco Ramirez, Mt. Sinai School of Medicine, New York, NY; Renato
Iozzo, Thomas Jefferson University, Philadelphia, PA, and Marian Young,
National Institute of Dental Research, respectively. For quantitation
of mRNA levels autoradiograms were scanned, analyzed using NIH
Image software, and normalized to the amount of 28 S RNA as revealed by
ethidium bromide staining.
Western Analysis--
Cell layers were harvested as for
proteoglycan analysis into 8 M urea buffer with protease
inhibitors, concentrated over DEAE-Sephacel, and precipitated with
ethanol. They were then digested with chondroitin ABC lyase or
heparitinase I and II, applied to SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Keene,
NH) using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA) for detection
of biglycan and decorin. Versican and perlecan-containing gels were
transferred using a Bio-Rad Transblot SD Semi-Dry Transfer Cell. The
transferred proteins were then detected with a series of primary
antibodies and enhanced chemiluminescence (Western-Light Chemiluminescent Detection System with CSPD substrate; Tropix, Bedford,
MA). Antibodies specific for biglycan (LF-51) and decorin (LF-136) (30)
were a gift from Larry Fisher, Bone Research Branch, NIDR, National
Institutes of Health, Bethesda, MD. Antibodies to versican (31) and
perlecan (R14, Ref. 32) were from, respectively, Richard LeBaron, The
University of Texas at San Antonio; and Gerardo Castillo, University of
Washington, Seattle, WA. Antibodies to TSG6 were the generous gift of
H-G Wisniewski of New York University (33).
Proteoglycan Aggregation--
Medium containing
[35S]sulfate-labeled proteoglycans from cells exposed to
4% strain for 48 h, or controls, was changed into an associative
buffer (500 mM sodium acetate, 0.25% CHAPS detergent, pH
5.8) by passage through a 0.5 × 12 cm size exclusion column of
Sephadex G50 (Amersham Pharmacia Biotech). The void peak was taken to
eliminate free [35S]sulfate, split into two aliquots and
applied to a 0.6 × 50 cm Sepharose CL-2B (Amersham Pharmacia
Biotech) molecular sieve column in the same associative buffer. Prior
to chromatography, one of the two aliquots was digested with 2 units of
Streptomyces hyaluronidase (Sigma) at 37 °C for 24 h. Under associative conditions, proteoglycans containing aggregates
will elute in the void volume. Digestion of components of the
aggregate, for example by hyaluronidase, will shift the elution
position of the proteoglycans to a later fraction (34, 35).
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RESULTS |
Mechanical Strain Selectively Increases Proteoglycan mRNA
Expression--
Human ASMC were exposed to 4% strain for 0, 12, 24, 36, or 48 h prior to harvest of all samples at the same time, with
no media changes in the 48-h period. Northern analysis revealed a time-dependent increase in mRNA levels for versican,
biglycan, perlecan, and type I collagen (Fig.
1). The greatest increase occurred in
versican mRNA levels, reaching 3.2-fold at 48 h. Levels of
versican and biglycan mRNA were greatest at 48 h whereas those of perlecan were greatest at 24 h, indicating that the synthesis of different proteoglycans may be differentially regulated by strain.
In contrast, decorin mRNA dropped rapidly by 12 h after the
application of strain and remained low throughout the experiment. In
additional zero strain controls, we observed no significant changes in
gene expression during the 48 h of culture.

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Fig. 1.
Strain alters the mRNA expression of
extracellular matrix molecules. A, confluent ASMC were
maintained in 10% FCS during 48-h exposure to 4% strain. Cells were
harvested for Northern blot analysis at 12-h intervals, and mRNA
was hybridized to cDNAs for versican, perlecan, collagen type I,
biglycan, and decorin. Mitochondrial 28 S RNA was stained with ethidium
bromide to show total loading. B, autoradiograms were
scanned; levels of mRNA were normalized to ethidium bromide
staining and expressed as -fold increase over 0 h levels. The
order of the bars is versican, perlecan, collagen type I,
biglycan, and decorin. Increases were observed for every mRNA
examined except for decorin, which decreased. These data are from a
single experiment representative of two similar experiments.
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Strain Increases the Accumulation of
[35S]Sulfate-labeled Proteoglycans--
To investigate
the effect of strain on proteoglycan synthesis, ASMC were metabolically
labeled with [35S]sulfate for the last 12 h of 12, 24, 36, or 48 h of 4% strain. A modest increase in total
incorporation of [35S]sulfate into proteoglycans, as
determined by CPC precipitation, peaked at 24-36 h (47% increase,
p < 0.005; Fig.
2A). The increase in
proteoglycans was apparent in both the cell layer and the medium.

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Fig. 2.
Strain increases the accumulation of
proteoglycans by ASMC. A, confluent ASMC were subjected
to 4% strain for 48 h and metabolically labeled with
[35S]sulfate at 12-h intervals. Control dishes were
treated identically except for the absence of strain. They were then
harvested for determination of total incorporation of radiolabeled
sulfate into proteoglycans. Equal aliquots were assayed per dish.
Strain increased the accumulation of total
[35S]sulfate-labeled proteoglycans, with a peak at 24-36
h. B, incorporation of [35S]sulfate into
chondroitin/dermatan sulfate, or heparan sulfate was determined by
digestion with chondroitin ABC lyase. Maximal incorporation into
chondroitin/dermatan sulfate peaked at 24-36 h, whereas the peak for
heparan sulfate occurred at 12-24 h in the medium, and 36-48 h in the
cell layer.
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The chondroitin/dermatan, and heparan sulfate portions of total
radiolabeled proteoglycans were determined by chondroitin ABC lyase
digestion of the cell and medium fractions, followed by CPC
precipitation (Fig. 2B). The maximum increase in
radiolabeled chondroitin/dermatan sulfate synthesis was in the medium
at 36-48 h (62% increase, p < 0.05), whereas that of
the heparan sulfate proteoglycans in the medium was at 12-24 h (87%
increase, p < 0.005), consistent with the early
increase in perlecan mRNA levels (Fig. 1). In the cell layer,
heparan sulfate incorporation reached a maximum at 36-48 h (58%
increase, p < 0.05). Because the heparan sulfate
component of the medium was approximately twice that of the cell layer,
the total of cell and medium heparan sulfate peaked at 12-24 h.
Hydrodynamic Size of Proteoglycans--
To determine whether
strain altered the relative quantities or average size of the different
classes of proteoglycan synthesized by ASMC, radiolabeled proteoglycans
collected after 24-36 h strain were isolated by ion affinity exchange
and subjected to molecular sieve chromatography on Sepharose CL-2B. Two
major peaks eluted at Kav ~0.28 and ~0.67 in
the medium and 0.42 and 0.78 in the cell layer (Fig.
3). The major component of the large peak
is versican and the smaller peak is a mixture of biglycan and decorin (8). No major differences were seen in the elution positions of these
radiolabeled peaks between control or strain cultures. The relative
amount of radiolabeled material secreted into the medium was ~80%
greater in the larger, versican-containing peak, indicating increased
synthesis in response to strain. This is consistent with increased
levels of versican mRNA (Fig. 1).

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Fig. 3.
Average hydrodynamic size of accumulated
proteoglycans is not altered by strain but total radiolabeled versican
is increased. [35S]sulfate-labeled proteoglycans
were purified and concentrated by ion exchange and applied to Sepharose
CL-2B chromatography. An equal fraction of each preparation was loaded
on the column. The resulting profiles had the same approximate peak
Kav values as the controls indicating no net
change in hydrodynamic size. Top, medium; bottom,
cell layer.
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The absence of a change in hydrodynamic size in the versican peak
suggests that the increased accumulation of
[35S]sulfate-labeled material is a result of increased
synthesis of the proteoglycan core proteins and their GAG chains rather than elongation of GAG chains as has been observed for the synthesis of
these proteoglycans by ASMC when stimulated by cytokines (8). A small
shift in the Kav of the second peak in the
medium to ~0.63, indicating a greater hydrodynamic size, was probably
because of an increase in the ratio of biglycan to decorin in that
peak, because biglycan is a larger macromolecule (8). This is supported by the increase in mRNA and protein expression of biglycan by strain (Figs. 1 and 4) and the decrease
in mRNA and protein for decorin (Figs. 1 and 4).

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Fig. 4.
Accumulation of proteoglycan core
proteins is altered by strain in ASMC. A, SDS-PAGE
reveals reduced accumulation of the chondroitin/dermatan sulfate
proteoglycan, decorin. [35S]sulfate-labeled
proteoglycans, prepared as for Fig. 3 from control and strain-treated
ASMC, were applied to 4-12% gradient PAGE with a 3.5% stacking gel.
B, confluent cells were subjected to strain for 36 h or
kept as controls in identical conditions lacking strain. Medium and
cell layer were then harvested and subjected to Western analysis with
antibodies against versican, biglycan, decorin, and perlecan. Equal
fractions of the total proteoglycans per dish were loaded for
comparison between strain and control treatments. Different quantities
were used for cell layer and medium. Accumulation of the core proteins
of these proteoglycans was increased in response to strain in all cases
except decorin, which declined.
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Strain Reduces the Accumulation of
[35S]Sulfate-labeled Decorin--
Proteoglycans from
cells exposed to strain for 24-36 h were applied to 4-12% gradient
SDS-PAGE. Decorin and biglycan run as separate bands in the resolving
gel whereas versican remains in the stacking gel (8). No change in the
relative amount of biglycan could be detected by this method. On the
other hand, the relative intensity of the decorin band in the medium
was greatly decreased, to ~30% of the control level (Fig.
4A). This is consistent with reduced production of decorin
mRNA by cells exposed to strain (see Fig. 1).
Synthesis of Proteoglycan Core Proteins by ASMC Is Altered by
Strain--
Western analysis was performed on medium and cell layer
extracts from ASMC exposed to strain for 24-36 h (Fig. 4B).
Increases were observed in the levels of versican, biglycan, and
perlecan. Versican core protein was increased to 126% of control
levels in both medium and cell layer. Perlecan was increased to 144 and 413% of control levels in the medium and cell layers, respectively. The percent increase in biglycan levels could not be determined by
scanning, because detectable levels were only found in medium after
application of strain. In contrast, the level of decorin core protein
was reduced to 13 and 89% of control levels in medium and cell layer, respectively.
Strain Increases Expression of TSG6--
In DNA microarray
experiments with the Affymetrix HU6800 microarray that were ongoing at
the same time as these experiments, we found that the gene for the
hyaluronan-binding protein TSG6 was among a small set of genes
increased by 4% strain in ASMC. Northern analysis confirmed that
mRNA for TSG6 was reproducibly increased by strain (Fig.
5). Both TNF-
(10 ng/ml) and strain led to modest increases in a single band (~90 kDa) recognized by
anti-TSG6 antibody in a Western analysis under reducing conditions (data not shown). This corresponds to a size that is larger than the
deduced molecular mass of TSG6 (~35 kDa); in four separate experiments, the cell lysate contained no bands or a very faint band at
~35 kDa. Thus, although these findings are consistent with previous
observations (made with the identical antibody used in this study) that
secreted TSG6 can form larger stable complexes with other molecules
(33), the identification of these 90-kDa bands as TSG6 is
tentative.

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Fig. 5.
Strain induces the hyaluronan-binding protein
TSG6. Northern blot analysis of mRNA from ASMC exposed to no
strain or strain. The steady-state level of mRNA for TSG6 was
increased 4.0 ± 1.0-fold by strain (24 h).
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Strain Increases Proteoglycan Aggregation--
To determine
whether increased synthesis of versican was accompanied by increased
formation of high molecular weight versican-hyaluronan aggregates, we
examined the presence of native aggregates using size exclusion
chromatography under associative conditions (34, 35). The absence of
material at the void volume of a Sepharose CL-2B molecular sieve column
under associative conditions in the control medium proteoglycans
indicated that no aggregate formed (Fig.
6A). A void volume peak in
medium from strained cells (Fig. 6B) indicated the presence
of a macromolecular aggregate of hyaluronan and versican. Digestion of
the samples with Streptomyces hyaluronidase prior to
chromatography shifted all of the aggregate from strained cells to the
included volume of the column, whereas it had no effect on the column
profile obtained with control medium. These findings demonstrate that
mechanical strain induces proteoglycan aggregation with hyaluronan.

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Fig. 6.
Strain increases the aggregation of
proteoglycans to hyaluronan. Medium containing
[35S]sulfate-labeled proteoglycans from cells exposed to
strain for 48 h, or controls, was subjected to chromatography with
Sephadex G50. Prior to chromatography, one of two aliquots was digested
with 2 units of Streptomyces hyaluronidase (Sigma) at
37 °C for 24 h. Left panel, disintegrations per min
per fraction (DPM) in medium from control cells without
hyaluronidase ( ) and with hyaluronidase ( ). Right
panel, DPM in medium from mechanically-stimulated cells without
hyaluronidase ( ) and with hyaluronidase ( ). The presence of a
void volume peak in the strain cells that is eliminated by
hyaluronidase indicates that strain causes aggregation with
hyaluronan.
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DISCUSSION |
In this study, we found that small, highly controlled
biomechanical deformation induces the synthesis of specific vascular proteoglycans including versican, biglycan, and perlecan, whereas decorin expression was suppressed. We also found increased expression of the hyaluronan-binding protein TSG6. Furthermore, mechanical strain
increased proteoglycan aggregation, suggesting that the cellular
response to deformation is highly coordinated. Because DNA microarray
experiments demonstrate that relatively few genes are altered by
mechanical strain in these cells (15), these experiments highlight the
restricted and specific molecular regulation of ASMC by biomechanical stimuli.
Vascular smooth muscle cells perform a sentinel role in arterial
mechanics. Under disturbed conditions such as hypertension, the
vascular smooth muscle cell must sense the changes and respond appropriately. These responses include changes in the extracellular matrix, and prior studies have demonstrated that mechanical strain increases collagen and proteoglycan synthesis by these cells (36-38). However, the molecular specificity of the proteoglycan increase has not
been characterized previously. Because different proteoglycans can have
markedly varied biological roles, we anticipated that not all vascular
proteoglycans would be regulated similarly. These experiments
demonstrate that decorin is specifically decreased by strain. Because
decorin binds to collagen (39) and regulates collagen cross-linking
(40), the decrease in decorin could promote disorganization of collagen
and a loosening of ECM.
One of the key potential defenses of cells against excess deformation
is secretion of proteoglycans. Proteoglycans, through negatively
charged glycosaminoglycan chains, can serve as molecular sponges that
cushion against mechanical forces. The increase of versican-hyaluronan
complexes shown in this study may provide a matrix environment that
thickens intima and protects the cell against mechanical stimuli. This
is consistent with the response of chondrocytes to
deformation; chondrocytes increase aggregation of the large
proteoglycan aggrecan to hyaluronan following deformation, which
provides a highly hydrated mechanical environment around the cell
(41).
The mechanisms by which strain differentially regulates vascular
proteoglycan synthesis are unclear. Our studies indicate that although
remarkably few genes are induced by biomechanical stimulation of
vascular smooth muscle cells, these responses are reproducible in
different cell sources (15). Mechanical signals activate many different
signal transduction pathways, but currently no single transcriptional
regulatory element or combination of elements can explain cell-specific
mechanical responses.
Among the relatively restricted subset of genes induced by the
small biomechanical stimulus of 4% biaxial strain is TSG6. This
hyaluronan-linking protein is primarily known as a pro-inflammatory response gene that is induced by cytokines, but it is intensely overexpressed in mechanically injured arteries (42). The impact of
mechanical strain on hyaluronan synthesis will need to be explored to
address the hypothesis that increased aggregation may be because of
increased hyaluronan synthesis.
In some circumstances, excess accumulation of vascular proteoglycans
may be detrimental. Williams and Tabas proposed that retention of
atherogenic lipoproteins in the artery is a central process in
atherogenesis, a hypothesis known as the Response-to-Retention hypothesis (43). Proteoglycans may serve as the reservoir for arterial
lipoproteins through a specific association with the apo-B100 protein;
a single point mutation in the apo-B100 protein inhibits binding to
proteoglycans without affecting low density lipoprotein (LDL) receptor
binding (44). Immunohistochemical studies have shown that biglycan
colocalizes with apo-B in the atherosclerotic intima, and both versican
and biglycan can bind LDL (45). Thus, whereas accumulation of
proteoglycans may contribute to mechanical properties of the artery, it
may also provide a subendothelial retention reservoir for lipoproteins.
In this study, we demonstrate that biomechanical strain selectively
up-regulates expression of biglycan and versican, two vascular
proteoglycans that can serve as LDL binding reservoirs. Thus, enhanced
proteoglycan synthesis may serves as an initial defense against
mechanical stress that can nonetheless prove deleterious as LDL
infiltrates the vessel wall.