Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1004-1013

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Evanko, S. P.
Right arrow Articles by Wight, T. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Evanko, S. P.
Right arrow Articles by Wight, T. N.
Related Collections
Right arrow Pathophysiology
Right arrow Smooth muscle proliferation and differentiation
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1004-1013.)
© 1999 American Heart Association, Inc.

Original Contributions

Formation of Hyaluronan- and Versican-Rich Pericellular Matrix Is Required for Proliferation and Migration of Vascular Smooth Muscle Cells

Stephen P. Evanko; John C. Angello; Thomas N. Wight

From the Departments of Pathology (S.P.E., T.N.W.) and Biochemistry (J.C.A.), University of Washington, Seattle.

Correspondence to Thomas N. Wight, University of Washington School of Medicine, Department of Pathology, Box 357470, Seattle, WA 98195. E-mail tnw{at}u.washington.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red–positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.


Key Words: cell adhesion • atherosclerosis • mitosis • locomotion


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The accumulation of hyaluronan (HA) and proteoglycans (PGs) in the artery wall contributes to progression of atherosclerosis by trapping lipoproteins in the neointima and by imparting swelling properties to the tissue, thereby promoting stenosis.1 HA is a high molecular weight polysaccharide found in the extracellular matrix of many connective tissues. It has been shown to influence several biological processes including wound healing, angiogenesis, embryonic mesodermal condensation, tumor metastasis, and cell migration and proliferation.2 3 4 Experimental studies show that HA is increased dramatically around proliferating smooth muscle cells (SMCs) in the early stages of neointimal formation in balloon-injured rat carotid arteries.5 6 HA synthesis is upregulated in proliferating SMCs and fibroblasts in vitro.7 8 The HA receptor CD44 is also upregulated after injury in vivo, and has been linked to proliferative responses of SMCs to HA.9 There is also evidence that HA and its receptor, receptor for hyaluronan mediated motility (RHAMM), are important for locomotion of cultured SMCs.10 However, the mechanisms for the diverse effects of HA are not clear.

Versican is a major chondroitin sulfate PG (CSPG) produced by SMCs11 and has been shown to bind to HA.12 13 Versican synthesis is upregulated during SMC proliferation in vitro.14 Both HA and versican are major components of primary atherosclerotic plaques and the loose extracellular matrix of human restenotic lesions.5 15 16 17 18 Colocalization of HA and versican around SMCs in atherosclerotic plaques17 suggests that these molecules function together during processes involved in lesion formation. Although HA and versican are abundant in lesions of atherosclerosis, little is known regarding the capability of SMCs to organize these molecules in the pericellular matrix or regarding their functional role(s) during SMC proliferation and migration.

Therefore, we have examined the organization of HA and versican in the pericellular matrix of human SMCs in vitro, using a particle-exclusion assay, time-lapse video imaging, and electron microscopy. We show that formation of HA- and versican-rich pericellular matrices by SMCs is dynamic and rapid, and occurs preferentially during the detachment phase of cell migration and premitotic cell rounding. We also show that HA oligosaccharides, which block formation of the pericellular matrix and binding of HA to the cell surface, can inhibit proliferation and migration and alter cellular morphology.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reagents and Supplies
DMEM was purchased from Irvine Scientific. FBS was from Intergen Co. Falcon cell culture plasticware was from Becton-Dickinson. Platelet-derived growth factor-BB (PDGF-BB) was kindly provided by Dr Russell Ross, Department of Pathology, University of Washington. Centricon microconcentrators were from Amicon, Inc. Chamber slides were purchased from Nunc, Inc. Crystal Mount mounting medium was from Biomeda. Secondary antibody and peroxidase conjugates were from Zymed Laboratories, Inc. HA (from human umbilical cord, grade I), Streptomyces hyaluronidase, and other chemicals and reagents were from Sigma.

Cell Culture
Arterial SMCs were derived from the media of normal newborn human thoracic aortas by explant and were maintained in DMEM containing 10% FBS.19 These cells were kindly provided by Dr Russell Ross, Department of Pathology, University of Washington. Cells stained positively for SMC {alpha}-actin (data not shown) had the characteristic hill-and-valley morphology of cultured SMCs and were used between passages 7 and 14.

Visualization of Pericellular Matrix
Visualization and morphometric analysis of HA/versican matrices were done by using a particle-exclusion assay as previously described.20 With this assay, an otherwise invisible matrix that surrounds a number of cell types in vitro can be visualized by virtue of the steric exclusion properties of HA-PG gels. In brief, 1x105 cells were plated in 35-mm tissue culture dishes in DMEM containing 10% FBS. After 24 hours, 750 µL of a suspension of fixed and washed human erythrocytes ({approx}108/mL) was added to the cells and allowed to settle for 15 minutes. Pericellular matrix formation was quantitated from photographs by using a digitizing graphics pad to measure the area delimited by red blood cells and the area delimited by the cell membrane to give a coat-to-cell ratio.20 A ratio of 1.0 indicates no matrix. For quantitating the proportion of cells with a matrix, cells with a ratio of >=2.0 were considered to have pericellular matrices.

Cells were treated with HA-specific Streptomyces hyaluronidase at 1 U/mL of medium for 1 hour before visualization with the particle assay, to demonstrate the structural dependence of the pericellular matrix on HA. In some cases, the enzyme was added after the particles had settled, to view individual pericellular coats before and after digestion of HA (see Figure 1Down). To examine the effect of HA oligosaccharides on pericellular matrix formation, cells were incubated with the oligosaccharides at 30 µg of uronate/mL for 1 hour before addition of the red blood cells.



View larger version (183K):
[in this window]
[in a new window]
  		Figure 1. Human vascular SMCs organize HA-dependent pericellular matrices. A, The particle-exclusion assay was used to demonstrate the presence of the HA-dependent pericellular matrix, which was apparent as a clear zone between the SMCs and the red blood cells. B, The same field as in A, {approx}5 minutes after Streptomyces hyaluronidase was added to the culture dish. The hyaluronidase degraded the HA coats, allowing the red blood cells to contact the SMCs directly. C, Staining for HA, using a biotinylated HA-binding region of cartilage aggrecan. After fixation, the HA staining (outlined with a dashed line) was seen in a flocculent material that collapsed onto the culture dish and cell surface. Arrowheads indicate the leading edge of the lamellipodium. D, Staining for HA in cells treated with Streptomyces hyaluronidase before fixation. E, Immunostaining using a polyclonal antibody specific for versican. F, Normal IgG control. A and B, bar=50 µm. C through F, bar=15 µm.

The kinetics of pericellular matrix formation was observed by using time-lapse video microscopy in combination with the particle-exclusion method. Time-lapse video analysis was done by using a Nikon inverted microscope equipped with an extralong phase condenser and a heated incubator (Nikon, Inc). The cells were imaged through a video camera (Series 65; Dage-MTI, Inc) equipped with a high-resolution vidicon tube and connected to a time-lapse recorder (Model TLC2051R, Gyyr Products). The videotape was viewed on a monochrome monitor (Model 925; Lenco). HEPES (50 mmol/L) was added to buffer the medium. To evaluate the length of time required for pericellular matrix formation, cells with little or no matrix at the beginning of the assay were selected and monitored for 4 hours.

Preparation of Hyaluronan Fragments
Two hundred milligrams of HA was dissolved in 20 mL of 0.1 mol/L ammonium acetate, pH 6.0, and digested for 72 hours with 50 U of Streptomyces hyaluronidase. The enzyme was inactivated by boiling for 20 minutes and the HA fragments were fractionated by ultrafiltration through Centricon microconcentrators (Amicon) with molecular weight cutoffs of 50 kDa, followed by 3 kDa. HA fragments passing through the Centricon 50 but retained by the Centricon 3 are designated HA3–50k and fragments passing through the Centricon 3 are designated HAoligo. Analysis by chromatography on Biogel P4 (Bio-Rad) showed that the oligosaccharide fraction consisted primarily of hexasaccharides ({approx}45%) and tetrasaccharides ({approx}30%) with small amounts of octa-, deca-, and dodecasaccharides comprising the remainder. Samples were lyophilized, resuspended in PBS, and filter-sterilized. Uronic acid concentration was measured by the orcinol method.21 As a control to verify that the effect of the oligosaccharides was not caused by a low molecular weight contaminant in the HA preparation, disaccharides were prepared by exhaustive digestion of HA with chondroitin ABC lyase (ICN Pharmaceuticals), and filtration through Centricon 3 membranes as described above. This preparation consisted of 95% HA disaccharide, with only minor amounts of tetrasaccharides and hexasaccharides. High molecular weight HA was dissolved in PBS and sterilized by overnight exposure to UV light.

Growth and Migration Studies
For growth-inhibition studies with HA and HAoligo, SMCs were plated in 24-well plates in the presence of 10% FBS. On the following day, cells were switched to medium containing 1% human plasma derived serum for 2 days before addition of PDGF and HA fragments (above). Alternatively, HAoligo was added to cells maintained continuously in 10% FBS. Cells were harvested with trypsin at the appropriate times and counted with a Coulter counter. The effects of HA and HAoligo on SMC migration on collagen-coated membranes in response to PDGF was assessed by using a modified Boyden chamber.22 The number of cells that migrated to the underside of the membrane over a 4-hour period was determined by counting nuclei in 6 fields from each of 3 replicate wells. Data are expressed as number of cells per field. Statistical significance was evaluated with a Student's t test.

Immunocytochemistry
Cells were seeded in 4-well chamber slides and allowed to adhere for 24 hours. After rinsing with PBS, the cultures were fixed with 4% paraformaldehyde for 20 minutes at 22°C, then rinsed in PBS. To localize HA, cells were stained with biotinylated HA-binding region from cartilage PG at a dilution of 2 µg/mL. This probe specifically recognizes HA.23 Controls for HA staining included Streptomyces hyaluronidase digestion of the cells before fixation (4 U/mL in DMEM, 1 hour, 37°C) and preincubation of the biotinylated HA-binding region with 100 µg of HA. Rabbit polyclonal antibody specific for human versican, VC-E, was used at a 1:250 dilution, and was kindly provided by Dr Richard LeBaron (University of Texas at San Antonio).24 Normal rabbit IgG was used as a negative control. Antibodies and the biotinylated HA-binding region were applied in PBS containing 1% BSA for 1 hour at 22°C. After rinses, the versican antibody was detected by using a biotinylated rat anti-rabbit 2o antibody at a 1:1000 dilution. The samples were rinsed and then incubated with a 1:500 dilution of a streptavidin–horseradish–peroxidase conjugate. After rinses, color was developed for 10 minutes by using 3-amino-9-ethylcarbazole in 0.05 mol/L sodium acetate, pH 5.0. The substrate solution was prepared by dissolving 2 mg 3-amino-9-ethylcarbazole in 0.5 mL of DMSO and adding 10 mL of 0.05 mol/L sodium acetate, pH 5.0; 10 µL of 30% H2O2 was added just before use. Cells were mounted with Crystal Mount (Biomeda Inc).

Pericellular Matrix Ultrastructure
Cells grown on coverslips were rinsed in serum-free medium and fixed with Karnovsky's fixative in 0.1 mol/L sodium cacodylate, 2 mmol/L CaCl, and 5% sucrose, containing 0.2% ruthenium red25 (to precipitate PGs), for 1 hour at 22°C with or without prior digestion by Streptomyces hyaluronidase. Cells were rinsed with the cacodylate buffer containing 0.1% ruthenium red, and then postfixed in 1% OsO4 in cacodylate buffer, 0.05% ruthenium red for 1 hour at 22°C. Coverslips were rinsed gently by dipping several times in PBS, and then H2O. Cells were air-dried to maximize preservation of the fragile HA network, then coated for scanning electron microscopy. Other cultures were fixed as above, embedded in Epon, and examined by transmission electron microscopy.

HA Binding Assay
Fluorescein-labeled HA was prepared according to published methods.26 For the HA binding assay, SMCs were plated in 60-mm culture dishes and allowed to attach for 24 hours. Cells were rinsed with PBS and incubated with competitor (unlabeled high molecular weight HA, or HA fragments and oligosaccharides) at 300 µg of uronate/mL in 1.5 mL serum-free DMEM for 20 minutes at 4°C. Fluoresceinated HA (2 µg of uronate/mL) was then added and cells were incubated for 10 minutes at 4°C with rocking. Cells were then washed 3 times with cold PBS, and solubilized with 0.25 mol/L NaOH. Bound fluorescence was determined by using a fluorometer and compared with a standard curve of fluorescein-labeled HA.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Pericellular Matrix Formation by Human SMCs
A particle-exclusion assay27 was used to determine if human vascular SMCs were capable of producing and organizing HA-rich pericellular matrices. In this assay, the presence of a pericellular matrix can be demonstrated by the exclusion of fixed red blood cells from a clear, halo-like area adjacent to the cell (Figure 1AUp). Human SMCs elaborated and organized extensive pericellular matrices, which were removed entirely by digestion with Streptomyces hyaluronidase (Figure 1BUp). Under normal growth conditions in medium containing 10% FBS, {approx}40% of the cells displayed HA-dependent matrices.

HA was localized histochemically in these cultures by using a biotinylated protein (HA-binding region from bovine cartilage aggrecan) that specifically binds HA (Figure 1CUp). Staining for HA was positive in a flocculent material that localized to the cell surface and culture dish in a pattern similar to the pericellular gel outlined by the particle assay in living cells. Pericellular staining was most prominent in cells with a motile phenotype, ie, having a lamellipodium and a thin trailing uropod. Staining was usually concentrated around the trailing process. Thin cellular processes resembling retraction fibers were often seen extending into the areas where the HA-rich matrix was visible. Some staining was present in areas of membrane ruffling. Digestion of the cultures with Streptomyces hyaluronidase before fixation abolished virtually all of the pericellular staining (Figure 1DUp). However, there was prominent staining within vesicular structures that was not affected by hyaluronidase digestion. These results are consistent with recent observations of HA in caveolae in vascular cells in vivo.28 All staining in our studies was abolished by preincubation of the biotinylated HA-binding protein with an excess of HA, confirming its specificity for HA (data not shown).

Immunostaining with an antibody specific for versican was similar to HA staining, and was detected both in the flocculent material on the culture dish surrounding the cells and in material closely apposed to the cell surface on the main cell body (Figure 1EUp). Staining was also prominent on thin trailing processes. Digestion of the cultures with Streptomyces hyaluronidase removed most of the pericellular versican immunostaining (data not shown), thus confirming previous observations that CSPGs are tethered to cells via association with HA.29 30 31 Versican was not detected intracellularly under these conditions. No staining was seen with normal rabbit IgG (Figure 1FUp).

Cells grown on coverslips for 24 hours were fixed in the presence of ruthenium red to precipitate the PGs,25 air-dried to preserve the cell coat, and examined by scanning electron microscopy (Figure 2Down). Fine filaments (10 to 40 nm in thickness) extending away from the cell surface were prominent on the thin trailing processes (Figure 2ADown and 2BDown). The filaments often exceeded 10 µm in length and were decorated with ruthenium red granules, and were similar in size (50 to 200 nm) to the large PG granules visualized by transmission electron microscopy of the SMC extracellular matrix in vitro (Figure 2Down, inset) and in vivo.25 The strands and most of the granules were removed by treatment with Streptomyces hyaluronidase before fixation (Figure 2DDown). This is consistent with the abolishment of staining by the enzyme as described above, and suggests that these structures represent HA/versican aggregates.



View larger version (130K):
[in this window]
[in a new window]
  		Figure 2. Electron microscopy of the pericellular matrix. Cells grown on coverslips were fixed in the presence of ruthenium red to preserve PGs, air-dried, and examined by scanning electron microscopy. A, A thin cell process is shown with individual strands of putative HA, extending from the cell surface and associated ruthenium red–positive granules (arrows). B, A similar cell process with the strands of HA arranged in a dense, tangled network. Note that the PG granules are often seen at the intersection points of the putative HA strands. The inset shows a transmission electron micrograph of the ruthenium red granules in the SMC extracellular matrix and the interconnecting filaments. C, Dense bead-like clusters of ruthenium red granules (arrows) and filaments were seen on the surface of the main cell body in some cells. D, Digestion with Streptomyces hyaluronidase removes the fine filaments and the granules. Bar=1 µm, in all micrographs.

The thin filaments occurred either as individual strands (Figure 2AUp) or as a tangled network (Figure 2BUp). The ruthenium red–positive granules were often seen at intersection points in the network, suggesting that the PGs serve as points of entanglement for the strands of HA. In some cells, dense clusters of tightly "beaded" PG granules and HA filaments were seen on the cell surface (Figure 2CUp), in areas of membrane ruffling, and in the pericellular space (not shown). These results are consistent with earlier observations in chondrocytes,30 31 and suggest that the pericellular matrix gel of SMCs, which is made apparent by particle exclusion, forms as a result of the accumulation and entanglement of HA and aggregated PG in the pericellular space.

Association of HA/Versican Matrices With Motile and Mitotic Cells
On closer examination of pericellular matrices in live cultures, as shown by the particle-exclusion assay, SMCs with a flattened and spread morphology displayed very little if any surrounding matrix, and those with a motile morphology displayed prominent pericellular matrices (Figure 3ADown and 3BDown). The pericellular matrix was present at predictable places on locomoting SMCs, specifically around the trailing process, which was detached from the substrate and retracted. Very little or no matrix was seen at the leading lamellipodium except in areas of membrane ruffling, consistent with HA and versican staining (see Figure 1Up). This suggests that HA secretion or pericellular matrix organization may be directionally controlled in locomoting cells.



View larger version (230K):
[in this window]
[in a new window]
  		Figure 3. Organization of HA-dependent pericellular matrix occurs in migrating SMCs. A and B, Under normal growth conditions, SMCs, which were highly spread and flattened, displayed little or no pericellular matrix, whereas cells having a motile phenotype displayed well-organized cell coats. C, After scratch wounding of confluent monolayers, the cells migrating from the wound edge into the cleared area displayed large, well-defined pericellular matrices. Arrow indicates direction of migration from the edge of the monolayer. Scale, 1 red blood cell=8 µm.

In a model for cell migration, synthesis of HA was reported to be upregulated in SMC monolayers after scratch wounding.10 At 24 hours after wounding, the particle assay showed large deposits of pericellular matrix around and between cells at the wound edge (Figure 3CUp). Approximately 90% of the migrating and proliferating cells at the wound edge displayed prominent pericellular matrices. In contrast, only {approx}40% of the cells in sparse cultures display pericellular matrices under normal growth conditions. This suggests that active organization of the HA/versican-rich matrix is important for wound-induced migration.

Virtually all mitotic SMCs (identified by a rounded shape and clearly visible chromosomes) had an investment of pericellular matrix surrounding or immediately adjacent to them (Figure 4ADown and 4BDown). The thick, abundant matrix surrounding dividing cells stained intensely for HA and versican (Figure 4CDown and 4DDown). Figure 4DDown shows an example of a late telophase cell with abundant versican staining in the matrix in the cleavage furrow, suggesting that the matrix may facilitate separation of daughter cells.



View larger version (175K):
[in this window]
[in a new window]
  		Figure 4. HA- and versican-rich pericellular matrix is associated with mitotic SMCs. A and B, Examples of SMCs in different stages of mitosis with associated pericellular matrices. Virtually all of the mitotic cells under normal growth conditions displayed well-defined pericellular matrices. Scale, 1 red blood cell=8 µm. C, A cell in early telophase with positive HA staining, which appears as dark flocculent material on and around the cell. D, A cell in late telophase immunostained for versican. The dark reaction product was seen on the cell surface, in the cleavage furrow, and in the dense matrix around mitotic cells. Bar=15 µm.

Time-lapse video microscopy, in conjunction with the particle assay, was used to obtain information on the kinetics of pericellular matrix formation and its relation to cell migration and proliferation. These data provided clear evidence that HA/versican-rich matrix organization is a rapid process occurring in migrating and mitotic cells, and that there is a dynamic interdependence between pericellular matrix formation, cell shape, and detachment from the substrate (Figure 5Down). In as little as 14 minutes, a matrix coat several micrometers thick was organized around a trailing process, just before its retraction. Simultaneously, pericellular matrix formed around the main body of the cell as it detached and rounded up. Cell division was completed {approx}1 hour after pericellular matrix formation, after which the matrix appeared to dissolve into the medium. Cells that remained stationary during filming did not display any organized pericellular matrix. In motile cells, pericellular matrix appeared to form episodically at sites of membrane ruffling at the leading edge and continuously around the trailing process as the cell moved (Figure 5Down, lower right corner). Observations that some of the matrix moved with the cells and some was sloughed and left behind suggest that this is a highly malleable matrix that facilitates detachment and sliding of cellular processes across the substrate. In several other cases, a large investment of pericellular matrix appeared to impede cells from reestablishing firm adhesion to the plastic substrate. These data are consistent with the reported involvement of HA and versican in proliferating cells and with a proposed role for HA in cell detachment and rounding.2 7 32



View larger version (45K):
[in this window]
[in a new window]
  		Figure 5. Pericellular matrix formation is rapid and precedes mitosis. Time-lapse video microscopy sequence precisely shows the kinetics of pericellular matrix formation around SMCs. At the beginning of the sequence (13 hours:46 minutes:55 seconds), a cell with no visible pericellular matrix is pictured in the middle of the field with a thin, extended process (arrowhead) that is obscured by the red blood cells. Fourteen minutes later (14:00:59, second panel), as the cell starts to round up, a distinct pericellular matrix has formed around the cell process, pushing the red blood cells away. The process is detached from the substrate in the third panel, and is fully retracted by 14:17:02 (fourth panel). The cell completes mitosis 1 hour later (fifth panel). Also note another cell migrating toward the lower right corner, with a matrix forming along the sides and a trailing process.

HA Oligosaccharides Inhibit Proliferation and Migration of SMCs
It has been previously shown that a hexasaccharide was the smallest fragment of HA capable of binding to cell surface receptors33 and that HA hexasaccharides were effective at preventing formation of HA-dependent pericellular matrices in chondrocytes and also in other cells.20 Therefore, we used a preparation of small HA oligosaccharides (HAoligo) to determine if disruption of the association of the pericellular matrix with the cell surface could affect cell proliferation or migration. Cells treated with HAoligo did not display pericellular matrices (Figure 6Down). The HAoligo competed efficiently for binding of fluorescein-labeled HA to the cell surface (Figure 6Down).



View larger version (110K):
[in this window]
[in a new window]
  		Figure 6. Hyaluronan oligosaccharides inhibit pericellular matrix formation and HA binding. Cells were incubated for 60 minutes in the absence (A) or presence (B) of HAoligo (30 µg/mL) and then examined with the particle-exclusion assay. C, Matrix/cell area ratios were used to determine the proportion of cells with a pericellular matrix. D, Binding of fluorescein-labeled HA (fHA) in the absence or presence of a 300-fold excess of HA or HAoligo. Mean±SD, n=3.

In cell proliferation assays, HAoligo significantly inhibited cell growth as measured by cell number (Figure 7ADown). In contrast, slightly larger fragments of HA (HA3–50k) and high molecular weight HA had no inhibitory effect, or even a slight stimulatory effect, on cell growth. The inhibitory effect of HAoligo was dose dependent (Figure 7BDown), and occurred after PDGF stimulation of serum-starved, quiescent cultures (Figure 7CDown) and in cells cultured continuously in the presence of serum (Figure 7DDown). There was no decrease in cell number in quiescent cells treated with HAoligo, suggesting that this was not a cytotoxic effect (Figure 7CDown). The release of lactate dehydrogenase into the medium was not altered by treatment with HAoligo, also confirming that this was not a cytotoxic effect (data not shown).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 7. Hyaluronan oligosaccharides inhibit proliferation of human SMC. A, Cell number after 6 days of growth in medium containing 10% FBS in the presence or absence of 30 µg uronate/mL HAoligo, HA fragments 3 to 50 kDa in size (HA 3–50K), or high molecular weight HA. (B) Dose–response of growth inhibition by HAoligo in the presence of 10% FBS. C, Cells were made quiescent (QUIESC.) by incubation with 1% human plasma derived serum for 48 hours and then stimulated with 10 ng/mL PDGF in the absence or presence of 30 µg/mL HAoligo. D, Growth over a 9-day period in medium containing 10% FBS was inhibited by 30 µg/mL HAoligo. All samples were measured in triplicate and data represent mean±SD values.

The lack of an effect by high molecular weight HA suggests that the growth inhibition was not caused by a contaminant in the commercial HA preparation. To further verify that there was not a low molecular weight contaminant with antiproliferative activity in the HA preparation, disaccharides of HA were prepared by digestion of HA with chondroitinase ABC and fractionated by the same procedure used for the oligosaccharides. The HA disaccharides had no effect on cell growth (data not shown). Furthermore, protease treatment of the HAoligo preparation did not alter the inhibitory activity (data not shown).

After >=2 days of incubation in the presence of the HA oligosaccharides, clear morphological changes in the cells became apparent. Control cells typically displayed an elongated, spindle-shaped morphology and cells treated with HAoligo became flattened and highly spread, with prominent stress fibers apparent even under phase-contrast microscopy (Figure 8ADown and 8BDown). Area measurements confirmed that HAoligo altered cell shape (Figure 8CDown), suggesting that the presence of the pericellular matrix at the cell surface is important for SMCs to maintain their fusiform morphology in vitro.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 8. HA oligosaccharides alter cell shape. Cells were cultured for 4 days in medium containing 10% FBS only (A) or with 30 µg/mL of added HAoligo (B). HAoligo-treated cells became much more flattened and spread, and contained prominent stress fibers visible by phase-contrast microscopy (arrow) Bar=50 µm. C, Cell area measurements. Individual measurements and the mean±95% confidence limit values are shown (n=50).

As described above, pericellular matrix formation was correlated preferentially with detachment and sliding of the trailing uropod during cell locomotion, as observed by time-lapse video microscopy. Therefore, we examined the effect of disruption of pericellular matrix formation by HAoligo on PDGF-induced cell migration by using a modified Boyden chamber assay. HAoligo inhibited migration on collagen-coated membranes in response to PDGF in a dose-dependent manner (Figure 9Down). In contrast, similar concentrations of larger HA fragments or high molecular weight HA did not have a significant effect.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 9. HA oligosaccharides inhibit migration of SMCs. Migration of cells on collagen-coated membranes in response to 10 ng/mL PDGF-BB in the absence or presence of 30 µg/mL of either HA, HA fragments (3 to 50 kDa), or HAoligo over a 4-hour period was measured by using a modified Boyden chamber (mean±SD, n=3).

It was also observed in freshly plated cultures that most of the cells tended to elongate uniaxially into the characteristic motile shape whereas some cells tended to spread out radially and uniformly, maintaining a more flattened appearance. The particle-exclusion assay showed that the pericellular matrix formed around extending filopodia in the cells that tended to elongate uniaxially after plating, suggesting that the matrix may also facilitate extension of cellular processes along the substrate (Figure 10ADown). Treatment of freshly plated SMCs with HAoligo significantly inhibited the elongation ("spreading") of the cells into their characteristic spindle shape (Figure 10BDown).



View larger version (74K):
[in this window]
[in a new window]
  		Figure 10. HA oligosaccharides inhibit elongation of freshly plated SMCs. A, Two examples of pericellular matrix formation around extending filopodia (arrow) in freshly plated cells. This occurred particularly in cells that tended to elongate uniaxially into the characteristic spindle shape. The particle-exclusion assay was done 1 to 4 hours after plating. Scale, 1 red blood cell=8 µm. B, HAoligo (30 µg/mL) was added to cells at the time of plating and the effect on cell elongation ("spreading") was measured after 5 hours. Individual measurements and the mean±95% confidence limit values are shown.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study we have shown that migrating and proliferating human SMCs organize pericellular matrices rich in HA and the aggregating CSPG versican. We have also demonstrated, using time-lapse video microscopy, that pericellular matrix formation is a dynamic, active process that occurs preferentially during the detachment phase of migration and mitotic cell rounding. Interference with the binding of HA to the cell surface by small oligosaccharides inhibited proliferation and migration and altered cell morphology to a more flattened and spread appearance.

Our observations of pericellular matrix formation and its relation to SMC proliferation and migration in vitro are consistent with observations of atherosclerotic lesion formation in vivo. For example, colocalization of HA with proliferating cells after balloon injury of rat carotid arteries5 and double-injured rabbit arteries (T.N. Wight, unpublished data, 1998). correlates well with our observations of HA/versican-rich pericellular matrix formation around mitotic cells and in migrating cells after wounding in vitro (present study and that by Savani et al10 ). Furthermore, HA and versican colocalize around SMCs in both early and advanced lesions of primary atherosclerosis in nonhuman primates,17 and in the loose, myxoid extracellular matrix of human restenotic arteries.5 16 Although these molecules are found in the media of normal vessels, the staining is usually patchy and much less intense than in developing lesions.17 These observations suggest that formation of HA/versican-rich pericellular matrices in SMCs is crucial for processes leading to lesion formation.

There is evidence that HA can regulate cell growth and migratory activity in SMCs and other cells.9 10 34 35 36 37 Our results disagree with a previous study in which bovine aortic SMCs did not respond to HA oligosaccharides, but in bovine endothelial cells, DNA synthesis was stimulated.34 This may be because of differences between species, the amounts of endogenously produced HA or HA-binding molecules, or other experimental variables. We show that rapid, active elaboration of the HA/versican-rich matrix at the cell surface occurs at a specific time in motile cells and in premitotic SMCs, ie, just before detachment and/or rounding. This extends previous observations in fibroblasts that HA synthesis was high during cytokinesis and was necessary for cell detachment and mitosis.7 This is significant because detachment and mitotic cell rounding would be expected to take place during G2/M, and it has been shown that exogenous, soluble, recombinant RHAMM blocks ras-transformed fibroblasts in the G2/M phase of the cell cycle, possibly by competition for binding of HA to cell surface receptors.36 The inhibition of growth and morphological changes in response to HA oligosaccharides suggests that the presence of high molecular weight HA or, perhaps, the entire organized pericellular matrix at the cell surface is required for progression through the cell cycle. It is possible that the HA oligosaccharides affect HA receptor–mediated signaling, in view of recent evidence for regulation of mitogen-activated protein kinase activity and other intracellular signaling events by RHAMM38 and CD44.39 40 41 42 CD44 is known to be important for pericellular matrix formation in other cells,20 31 and dimerization and clustering of CD44 variants reportedly increases hyaluronate-binding capacity.43 In our experiments, only the small HA oligosaccharides (<3000 Da) were effective inhibitors of growth and migration whereas larger fragments (>3000 Da) were ineffective. One possible explanation that we are currently investigating is that the smaller HA fragments may prevent dimerization and clustering of CD44 and that this plays a role in SMC proliferation.

Time-lapse video microscopy allowed us to view the relation between pericellular matrix formation and cell behavior in individual cells. These data provide compelling evidence that one function of the HA/versican-rich pericellular matrix is to destabilize cellular attachment to the substrate during migration and rounding. The HA-dependent pericellular matrix may also help maintain the fusiform morphology in vitro. HA and versican (or related CSPGs) have been reported to have both adhesive and antiadhesive properties.32 44 45 46 Thus, the mechanism(s) by which HA or CSPGs mediate their effects on adhesion or migration remain speculative. However, one proposed mechanism by which this matrix may mediate cell detachment is based on the steric exclusion properties of HA-PG gels.2 30 This is the basis of the particle-exclusion assay, and was clearly evident in the rapid expulsion of the red blood cells away from the SMCs during matrix formation, and the subsequent detachment of the cells from the substrate. This property may also be important in regulating cell–cell contact, such as during limb morphogenesis where an HA-rich extracellular matrix can regulate mesenchymal cell condensation,2 47 or in facilitating separation of daughter cells after division (Tammi and Tammi48 and the present study). A role for HA in cell detachment is also supported by the finding that loosely adherent Chinese hamster ovary cell variants, isolated on the basis of ease of detachment, exhibited higher levels of HA synthesis compared with more tightly adherent variants,49 and that exogenous HA stimulated detachment of BALB 3T3 cells.32 Previous immunocytochemical studies have demonstrated in cultured fibroblasts that HA, versican, and CD44 are localized over the entire cell surface and concentrated in areas of membrane ruffling and in the perinuclear vicinity, but were excluded from focal contacts, suggestive of a role for these molecules in "de-adhesion."50 51 Our results are entirely consistent with these observations and indicate that actual organization of the matrix at the cell surface is important for this function. These studies suggest that HA and versican are essential by-products, inseparable from and necessary for specific events during SMC migration or proliferation, ie, cell detachment. It is noteworthy that a preexisting HA- and versican-rich matrix may not be required because the cells appear to elaborate and use it simultaneously. We speculate that the relative amount and material properties of the nonfibrillar matrix in the pericellular milieu plays a role in the ability of cells to detach from each other and from more adhesive matrix components, to round up or migrate in vivo.

Tight control of the physical mucoid properties of the pericellular matrix may be an important regulatory mechanism for SMCs during atherogenesis or the response to injury and could affect HA receptor signaling. HA is capable of self-association, and forms networks even at low concentrations.52 The length of HA may be critical for the integrity, viscosity, and malleability of the HA network. Inclusion of an aggregating CSPG could act to increase the viscosity of the gel,53 54 and add swelling pressure to the matrix because of the CS chains.20 30 CSPGs also decrease the permeability of the pericellular coat.55 CSPGs can potentially stiffen the HA network because of the shortening of HA, which results from the interaction during aggregate formation,56 enhancing the steric exclusion property. This could influence organization of other matrix components, or directly modify the effect of HA on cell behavior by altering cell/matrix tensegrity and, in turn, mechanically coupled signaling.57 These ideas may relate to previous studies showing inhibition of neural crest cell migration in response to exogenous CSPGs, thought to be mediated by interaction with cell surface HA.58

The idea that aggregate formation may occur in atherosclerotic lesions and that HA and versican function together is supported by electron microscopic studies of the extracellular matrix of lesions that show abundant ruthenium red–positive PG granules interconnected by a network of fine filaments directly adjacent to SMCs.25 As shown ultrastructurally in the present study, the network of hyaluronidase-sensitive filaments and PG granules surrounding SMCs in vitro is consistent with this, and is highly similar to endogenous pericellular matrices and those reconstituted from exogenous CSPG and HA in chondrocytes.31 It is clear that the structure of the pericellular matrix is dependent on HA.2 3 27 Furthermore, it has also been shown that aggregating CSPGs are required to visualize an expanded matrix by particle exclusion or other assays,20 30 although it is likely that other molecules are present. This suggests that the relative amounts of HA, versican, and stabilizers such as link protein, and the degree of aggregate formation, may be important for the general stability and material properties of the vascular extracellular matrix in vivo, as has been demonstrated for HA-PG aggregates in articular cartilage.59

An HA/CSPG-rich pericellular matrix could function to mediate cell detachment from other matrix components caused by the steric exclusion properties, and simultaneously facilitate cell rounding because of its apparently high malleability, which we have noted in the present study. Cells plated on highly malleable substrates tend to round up because of the traction forces that they place on the surrounding matrix.60 Relative to the tension-bearing elements of the extracellular matrix such as collagen fibrils, the nonfibrillar components are presumably much more malleable, providing less resistance to cellular traction forces, and therefore have direct bearing on cell shape and mechanically coupled signaling.61 Thus, in addition to integrin inactivation,22 HA/versican pericellular matrix formation potentially serves as another important, but infrequently considered, mechanism for controlling and counterbalancing the traction aspect of cell locomotion or other processes through this "lubricative" mechanism and the simultaneous steric interference with adhesion to fibrous matrix components. These findings may have important ramifications regarding the cell/matrix interface, adhesion and cell shape–related signaling events, and in developing treatments for atherosclerosis and restenosis.


*    Acknowledgments
 
This work was supported in part by a grant from the National Institutes of Health (HL-18645 to T.N.W.). S.P.E. was supported by a fellowship from the American Heart Association, Washington. The authors would like to thank the following people for their generosity: Richard LeBaron (Division of Life Sciences, University of Texas at San Antonio) for providing the versican-specific antibody, Charles Underhill (Department of Anatomy and Cell Biology, Georgetown University) for the biotinylated hyaluronan binding proteoglycan, Russell Ross (Department of Pathology, University of Washington) for the PDGF and human SMC; and Stephanie Lara for assistance with electron microscopy.

Received August 3, 1998; accepted September 21, 1998.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Wight TN. The vascular extracellular matrix. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven Publishers, 1996:421–440.
  2. Toole BP. Developmental role of hyaluronate. Connect Tissue Res. 1982;10:93–100.[Medline] [Order article via Infotrieve]
  3. Laurent TC, Fraser JRF. Hyaluronan. FASEB J.. 1992;6:2397–2404.[Abstract]
  4. Knudson CB, Knudson W. Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J. 1993;7:1233–1241.[Abstract]
  5. Riessen R, Wight TN, Pastore C, Henley C, Isner JM. Distribution of hyaluronan during extracellular matrix remodeling in human restenotic arteries and balloon-injured rat carotid arteries. Circulation. 1996;93:1141–1147.[Abstract/Free Full Text]
  6. Chajara A, Levesque H, Courel M-N, Chauzy C, Maingonnat C, Bertrand P, Delpech B. Hyaluronan and hyaluronectin production in injured rat thoracic aorta. Atherosclerosis. 1996;125:193–207.[Medline] [Order article via Infotrieve]
  7. Brecht M, Mayer U, Schlosser E, Prehm P. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem J. 1986;239:445–450.[Medline] [Order article via Infotrieve]
  8. Papakonstantinou E, Karakiulakis G, Roth M, Block LH. Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1995;92:9881–9885.[Abstract/Free Full Text]
  9. Jain M, He Q, Lee W-S, Kashiki S, Foster LC, Tsai J-C, Lee M-E, Haber E. Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury. J Clin Invest. 1996;97:596–603.[Medline] [Order article via Infotrieve]
  10. Savani RC, Wang C, Yang B, Zhang S, Kinsella MG, Wight TN, Stern R, Nance DM, Turley EA. Migration of bovine aortic smooth muscle cells after wounding injury: the role of hyaluronan and RHAMM. J Clin Invest. 1995;95:1158–1168.
  11. Yao LY, Moody C, Schonherr E, Wight TN, Sandell LJ. Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization, and immunohistochemistry. Matrix Biol. 1994;14:213–225.[Medline] [Order article via Infotrieve]
  12. LeBaron RG, Zimmerman DR, Rouslahti E. Hyaluronate binding properties of versican. J Biol Chem. 1992;267:10003–10010.[Abstract/Free Full Text]
  13. Chang Y, Yanagashita M, Hascall VC, Wight TN. Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina) aorta. J Biol Chem. 1983;258:5679–5688.[Abstract/Free Full Text]
  14. Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN. Effects of platelet-derived growth factor and transforming growth factor-ß1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem. 1991;266:17640–17647.[Abstract/Free Full Text]
  15. Levesque H, Girard N, Maingonnat C, Delpech A, Chauzy C, Tayot J, Courtois H, Delpech B. Localization and solubilization of hyaluronan and of the hyaluronan-binding protein hyaluronectin in human normal and arteriosclerotic arterial walls. Atherosclerosis. 1994;105:51–62.[Medline] [Order article via Infotrieve]
  16. Wight TN, Lara S, Riessen R, LeBaron R, Isner J. Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am J Pathol. 1997;151:963–973.[Abstract]
  17. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics and the proximity of PDGF and TGF-ß. Am J Pathol. 1998;152:533–546.[Abstract]
  18. Gutierrez P, O'Brien KD, Ferguson M, Nikkari ST, Alpers CE, Wight TN. Differences in the distribution of versican, deocrin, and biglycan in atherosclerotic human coronary arteries. Cardiovasc Pathol. 1997;6:271–278.
  19. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. TGF-ß induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell. 1990;63:515–524.[Medline] [Order article via Infotrieve]
  20. Knudson W, Bartnik E, Knudson CB. Assembly of pericellular matrices by COS-7 cell transfected with CD44 lymphocyte-homing receptor genes. Proc Natl Acad Sci U S A. 1993;90:4003–4007.[Abstract/Free Full Text]
  21. Brown AH. Determination of pentose in the presence of large quantities of glucose. Arch Biochem Biophys. 1946;11:269–278.
  22. Koyama N, Seki J, Vergel S, Mattson EJR, Yednock T, Kovach NL, Harlan JM, Clowes AW. Regulation and function of an activation-dependent epitope of the ß1 integrins in vascular cells after balloon injury in baboon arteries and in vitro. Am J Pathol. 1996;148:749–761.[Abstract]
  23. Underhill CB, Nguyen H, Shizari M, Culty M. CD44 positive macrophages take up hyaluronan during lung development. Dev Biol. 1993;155:324–336.[Medline] [Order article via Infotrieve]
  24. du Cros DL, LeBaron RG, Couchman JR. Association of versican with dermal matrices and is potential role in hair follicle development and cycling. J Invest Dermatol. 1995;105:426–431.[Medline] [Order article via Infotrieve]
  25. Wight TN, Ross R. Proteoglycans in primate arteries. I. Ultrastructural localization and distribution in the intima. J Cell Biol. 1975;67:660–674.[Abstract/Free Full Text]
  26. DeBelder AN, Wik KO. Preparation and properties of fluorescein-labelled hyaluronate. Carbohydr. Res. 1975;44:251–257.[Medline] [Order article via Infotrieve]
  27. Clarris BJ, Fraser JRE. On the pericellular zone of some mammalian cells in vitro. Exp Cell Res. 1968;49:181–193.[Medline] [Order article via Infotrieve]
  28. Eggli PS, Graber W. Association of hyaluronan with rat vascular endothelial cells and smooth muscle cells. J Histochem Cytochem. 1995;43:689–697.[Abstract]
  29. Yeo T-K, Yeo K-T, Wight TN. Differential transport kinetics of chondroitin sulfate and dermatan sulfate proteoglycan by monkey aorta smooth muscle cells. Arch Biochem Biophys. 1992;294:9–16.[Medline] [Order article via Infotrieve]
  30. Lee GM, Johnstone B, Jacobson K, Caterson B. The dynamic structure of the pericellular matrix on living cells. J Cell Biol. 1993;123:1899–1907.[Abstract/Free Full Text]
  31. Knudson W, Aguiar DJ, Hua Q, Knudson CB. CD44-anchored hyaluronan-rich pericellular matrices: and ultrastructural and biochemical analysis. Exp Cell Res. 1996;228:216–228.[Medline] [Order article via Infotrieve]
  32. Abatangelo G, Cortivo R, Martelli M, Vecchia P. Cell detachment mediated by hyaluronic acid. Exp Cell Res. 1982;137:73–78.[Medline] [Order article via Infotrieve]
  33. Underhill CB, Toole BP. Binding of hyaluronate to the surface of cultured cells. J Cell Biol. 1979;82:475–484.[Abstract/Free Full Text]
  34. West DC, Kumar S. The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp Cell Res. 1989;183:179–196.[Medline] [Order article via Infotrieve]
  35. Mast BA, Diegelmann RF, Krummel TM, Cohen IK. Hyaluronic acid modulates proliferation, collagen, and protein synthesis of cultured fetal fibroblasts. Matrix. 1993;13:441–446.[Medline] [Order article via Infotrieve]
  36. Mohapatra S, Yang X, Wright JA, Turley EA, Greenberg AH. Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression. J Exp Med. 1996;183:1663–1668.[Abstract/Free Full Text]
  37. Hall CL, Wang C, Lange LA, Turley EA. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity. J Cell Biol. 1994;126:575–588.[Abstract/Free Full Text]
  38. Zhang S, Chang MCY, Zylka D, Turley S, Harrison R, Turley EA. The hyaluronan receptor RHAMM regulates extracellular-regulated kinase. J Biol Chem. 1998;273:11342–11348.[Abstract/Free Full Text]
  39. Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC. Interaction between the adhesion receptor, CD44 and the oncogene product, p185HER2, promotes human ovarian tumor cell activation. J Biol Chem. 1997;272:27913–27918.[Abstract/Free Full Text]
  40. Ilangumaran S, Briol A, Hoessli DC. CD44 selectively associates with active src family protein tyrosine kinases lck and fyn in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes. Blood. 1998;91:3901–3908.[Abstract/Free Full Text]
  41. Rao CM, Deb TB, Datta K. Hyaluronic acid induced hyaluronic acid binding protein phosphorylation and inositol triphosphate formation in lymphocytes. Biochem Mol Biol Int. 1996;40:327–337.[Medline] [Order article via Infotrieve]
  42. Taher TE, Smit L, Griffionen AW, Schilder-Tol EJ, Borst J, Pals ST. Signalling through CD44 is mediated by tyrosine kinases. J Biol Chem. 1996;271:2863–2867.[Abstract/Free Full Text]
  43. Sleeman J, Rudy W, Hofmann M, Moll J, Herrlich P, Ponta H. Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity. J Cell Biol. 1996;135:1139–1150.[Abstract/Free Full Text]
  44. Orlidge A, D'Amore PA. Cell specific effects of glycosaminoglycans on the attachment and proliferation of vascular wall components. Microvasc Res. 1986;31:41–53.[Medline] [Order article via Infotrieve]
  45. Yamagata M, Kimata K. Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican. J Cell Sci. 1994;107:2581–2590.[Abstract]
  46. Ernst H, Zanin MKB, Everman D, Hoffman S. Receptor-mediated adhesive and anti-adhesive functions of chondroitin sulfate proteoglycan preparations from embryonic chicken brain. J Cell Sci. 1995;108:3807–3816.[Abstract]
  47. Knudson CB, Munaim SI, Toole BP. Ectodermal stimulation of the production of hyaluronan-dependent pericellular matrix by embryonic limb mesodermal cells. Dev Dyn. 1995;204:186–191.[Medline] [Order article via Infotrieve]
  48. Tammi R, Tammi M. Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes. Exp Cell Res. 1991;195:524–527.[Medline] [Order article via Infotrieve]
  49. Barnhart BJ, Cox SH, Kraemer PM. Detachment variants of Chinese hamster cells: hyaluronic acid as a modulator of cell detachment. Exp Cell Res. 1979;119:327–332.[Medline] [Order article via Infotrieve]
  50. Yamagata M, Saga S, Kato M, Bernfield M, Kimata K. Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts: implications for their roles in cell-substratum adhesion. J Cell Sci. 1993;106:55–65.[Abstract]
  51. Turley EA, Torrance J. Localization of hyaluronate and hyaluronate-binding protein on motile and non-motile fibroblasts. Exp Cell Res. 1984;161:17–28.
  52. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation: hyaluronan is a very efficient network forming polymer. Biochem J. 1991;274:699–705.
  53. Mow VC, Mak AF, Lai WM, Rosenberg LC, Tang L-H. Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations. J Biomech. 1984;17:325–338.[Medline] [Order article via Infotrieve]
  54. Soby L, Jamieson AM, Blackwell J, Choi HU, Rosenberg LC. Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate. Biopolymers. 1990;29:1587–1592.[Medline] [Order article via Infotrieve]
  55. Heldin P, Suzuki M, Teder P, Pertoft H. Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan containing coat around normal human mesothelial cells. J Cell Physiol. 1995;165:54–61.[Medline] [Order article via Infotrieve]
  56. Morgelin M, Paulsson M, Heinegard D, Aebi U, Engel J. Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates. Biochem J. 1995;307:595–601.
  57. Ingber DE. Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci. 1993;104:613–627.[Medline] [Order article via Infotrieve]
  58. Perris R, Perissinotto D, Pettway Z, Bronner-Fraser M. Inhibitory effects of PG H-aggrecan and PG M-versican on avian neural crest cell migration. FASEB J. 1996;10:293–301.[Abstract]
  59. Lohmander LS. The release of aggrecan fragments into synovial fluid after joint injury and in osteoarthritis. J Rheumatol Suppl. 1995;43:75–77.[Medline] [Order article via Infotrieve]
  60. Vernon RB, Angello JC, Iruela-Arispe ML, Lane TF, Sage EH. Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab Invest. 1992;66:536–547.[Medline] [Order article via Infotrieve]
  61. Chicurel ME, Singer RH, Meyer CJ, Ingber DE. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions. Nature. 1998;392:730–733.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Meran, D. W. Thomas, P. Stephens, S. Enoch, J. Martin, R. Steadman, and A. O. Phillips
Hyaluronan Facilitates Transforming Growth Factor-{beta}1-mediated Fibroblast Proliferation
J. Biol. Chem., March 7, 2008; 283(10): 6530 - 6545.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. Kastner, G. J. Thomas, R. H. Jenkins, M. Davies, and R. Steadman
Hyaluronan Induces the Selective Accumulation of Matrix- and Cell-Associated Proteoglycans by Mesangial Cells
Am. J. Pathol., December 1, 2007; 171(6): 1811 - 1821.
[Abstract] [Full Text] [PDF]