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Circulation Research. 2001;88:77-83

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(Circulation Research. 2001;88:77.)
© 2001 American Heart Association, Inc.

Cellular Biology

Hyaluronan Enhances Contraction of Collagen by Smooth Muscle Cells and Adventitial Fibroblasts

Role of CD44 and Implications for Constrictive Remodeling

Jeffrey A. Travis, Michael G. Hughes, James M. Wong, William D. Wagner, Randolph L. Geary

From the Departments of Surgery (J.A.T., M.G.H., J.M.W., R.L.G.) and Pathology, Section on Comparative Medicine (W.D.W., R.L.G.), Wake Forest University School of Medicine, Winston-Salem, NC.

Correspondence to Randolph L. Geary, MD, FACS, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157. E-mail rgeary{at}wfubmc.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone {approx}65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7.7±0.9; 2%, 7.1±0.7; 10%, 6.7±0.5; 50%, 5.6±0.9; P<0.05 for >=10%), cell spreading and telopodia, and pericellular accumulation of collagen fibrils. These effects were mediated in part by cellular HA binding, because an antibody against CD44 receptors blocked pericellular collagen accumulation and enhanced gel contraction without altering cell shape. The role of CD44 was specific, because inhibiting receptor for hyaluronic acid–mediated motility (RHAMM) had no effect. Blocking ß1-integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and ß1-integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.


Key Words: hyaluronan • smooth muscle cells • adventitial fibroblasts • restenosis • remodeling


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Remodeling contributes significantly to restenosis when reorganization of cells and matrix at sites of injury leads to shrinkage of the artery wall.1 2 This constrictive remodeling may be analogous to wound healing, where fibrosis and wound contraction help close tissue defects.3 4 Wound contraction results from adhesive interactions between myofibroblasts and wound matrix, and the matrix composition can significantly alter the extent of contraction.4 5 Collagen and the glycosaminoglycan hyaluronan (HA) are prominent components of both wound matrix and new matrix elaborated within the artery wall at sites of angioplasty.3 5 6 HA has been associated with improved wound repair and a lack of fetal scarring,5 but its role in the artery wall response to injury is poorly defined.

HA is a large (>106 kDa), negatively charged linear polysaccharide of repeating N-acetyl glucosamine and glucuronic acid dimers. Its size, charge, and structure impart unique chemical and viscoelastic properties to tissues, creating a loose, hygroscopic extracellular environment conducive to cell movement and tissue remodeling.7 HA binds to specific cell-surface receptors, including CD44 and receptor for hyaluronic acid–mediated motility (RHAMM), and to other matrix molecules, such as collagen and proteoglycans.7 8 9 10 Fetal tissues are particularly rich in HA,7 11 but content falls during development,7 and in the mature artery wall, small amounts persist most prominently in the adventitia and subendothelial space.3 6 12 HA later accumulates in the artery wall in response to injury and inflammation and thus is prominent in the atherosclerotic intima and sites of angioplasty.3 6 12 13 Although HA exhibits significant effects on vascular cell growth and migration in culture,10 13 14 15 few studies have explored its effects on the arterial response to injury in vivo16 and none have addressed the impact of HA on remodeling of extracellular matrix by smooth muscle cells and adventitial fibroblasts.

We hypothesized that altering the balance between HA and other matrix components prominent at sites of arterial injury would affect remodeling, speculating that increasing HA would inhibit matrix contraction and wall shrinkage. An in vitro assay of matrix contraction was used to determine whether HA would inhibit collagen remodeling by aortic adventitial fibroblasts and smooth muscle cells. Unexpectedly, an opposite effect was demonstrated. HA significantly increased matrix contraction by both smooth muscle cells and adventitial fibroblasts through a mechanism of enhanced collagen reorganization that was dependent on CD44-receptor ligation. These observations are consistent with an important role for HA in regulating matrix reorganization and constrictive remodeling of the healing artery wall. Cellular HA interactions provide a potential target for antirestenosis therapies aimed at improving remodeling.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
Primary cultures of medial smooth muscle cells and adventitial fibroblasts were obtained from cynomolgus monkey aorta. Vessels were opened longitudinally and gently scraped to remove endothelia. Adventitia and media were separated, digested in collagenase A (5 mg/mL, Sigma), and plated in 6-cm dishes with complete medium (DMEM, 100 IU/mL Pen/Strep, and 10% FCS) at 37°C and 5% CO2. Cells were passaged when nearly confluent and studied between passages 3 and 7. Smooth muscle cells were characterized by {alpha}-actin staining and hill-and-valley growth, whereas fibroblasts exhibited typical spindle morphology and less {alpha}-actin staining. Both stained negatively for von Willebrand factor.

Collagen Gel Contraction Assay
Collagen gels were prepared as described by Lee et al,17 with minor modifications. Twenty-four well plates were precoated with 1% agarose (FMC BioProducts) to promote gel detachment.18 Type I collagen (Collagen Corp) was diluted with complete medium to 2 mg/mL and adjusted to pH 7.4. Smooth muscle cells or fibroblasts were trypsinized when 80% confluent and resuspended at 6x105 cells/mL. Equal volumes of collagen and cells were then combined to give 1 mg collagen and 3x105 cells per mL and 500 µL pipetted into each well. Gels were polymerized at 37°C, and diameters were recorded over 24 hours. Contraction was measured relative to the initial gel diameter of 18 mm. Experiments comparing contraction of collagen gels to those with HA (see below) were then standardized at 18 hours. All assays were repeated 3 times with triplicate wells per experimental condition.

Effects of HA and Inhibition of ß1-Integrins, CD44, RHAMM, and Metalloproteinases
Gels were constructed as above, but high molecular weight HA (Healon, 2 mg/mL, Pharmacia) was added to collagen (2 mg/mL) to give final concentrations of 0% (control), 2%, 10%, 25%, or 50% (wt/wt). Matrix solutions were each combined with an equal volume of cells and polymerized, and gel diameters were measured at 18 hours.

After establishing the effect of HA on gel contraction, the role of cell adhesion to collagen via ß1-integrins and to HA via CD44 and RHAMM receptors was assessed using blocking antibodies. Smooth muscle cells and fibroblasts were prepared as above and pretreated for 30 minutes with increasing concentrations (0.5 to 50 µg/mL) of antibody specific for the ß1-integrin (mAb13a, a generous gift from Dr Kenneth Yamada, National Institutes of Health/National Institute of Dental and Craniofacial Research, Bethesda, Md), CD44 receptor (clone KM114, PharMingen),19 or RHAMM receptor (clone HV41.3, a generous gift from Dr Eva Turley, Hospital for Sick Children, Toronto, Ontario, Canada).10 14 Cells were then combined with matrix and polymerized, and antibody effects on contraction were assessed at 18 hours. In each blocking experiment, additional cells were pretreated with equal concentrations of nonimmune IgG1 (PharMingen) to control for nonspecific effects.

To determine whether the effects of HA on collagen reorganization and gel contraction were attributable to altered collagenase activity, gels were treated with the broad-spectrum metalloproteinase (MMP) inhibitor batimastat (BB94, British Biotech Pharmaceuticals Ltd).20 Batimastat was dissolved in DMSO as suggested by the manufacturer and then diluted with PBS and added to gels at a final concentrations ranging from 1 to 10 µg/mL (<0.1% DMSO). Controls received the dilute vehicle alone. Gels were then polymerized, and diameter was measured at 18 hours.

Cell Density, Immunofluorescence, and Sirius Red Staining
Gels of collagen or 50% HA, with and without CD44 antibody (50 µg/mL), were allowed to contract for 18 hours and then fixed overnight in 4% paraformaldehyde. After paraffin embedding, adjacent sections were cut from the center of triplicate gels from each condition and stained with hematoxylin for cell counting or an FITC-labeled antibody against {alpha}-actin (Sigma) to define cell shape.3 Cell nuclei were counted in nonoverlapping fields at x400 using an eyepiece reticule and expressed as cells per mm2. Additional sections were cut from each gel and stained with Sirius red to label collagen as previously described.21 Sections were viewed with standard light microscopy and also polarized light to define changes in the distribution of collagen fibrils associated with varying gel composition.

Statistics
Assays were completed in triplicate, and gel diameter and cell number were reported as the mean±SD for each experiment. Results were compared among experimental conditions for each cell type using a 2-tailed Student’s t test with significance assigned at P=0.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Matrix Contraction
Both smooth muscle cells and adventitial fibroblasts contracted gels of collagen alone to 50% to 65% of starting diameter within 24 hours (P<0.001, Figure 1Down). As previously described, collagen gel contraction was mediated by adhesion to cell-surface ß1-integrins (collagen receptors), because pretreating cells with ß1-blocking antibody completely inhibited contraction (Figure 1Down). Nonimmune IgG had no effect on contraction over the same range of antibody concentrations for either cell type (data not shown). Collagen contraction was also dependent on cells and serum, because gels polymerized without cells or in the absence of serum showed no contraction over 24 hours.



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  		Figure 1. Change in diameter over time of collagen gels seeded with either aortic medial smooth muscle cells (SMCs) or adventitial fibroblasts (AFs). Each point represents the mean of triplicate gels±SD for a representative assay. Also shown is the inhibitory effect of pretreating smooth muscle cells with a blocking antibody specific for ß1-integrin collagen receptors. The effect was similar for fibroblasts, and the 18-hour time point was selected for subsequent experiments.

Adding HA to collagen gels failed to inhibit contraction as hypothesized. Instead, the degree of contraction increased with the concentration of HA (Figure 2Down), and the effect was the same for both cell types. Increased contraction of gels containing HA was not attributable to a steric effect of increasing the ratio of cells to collagen, because HA also increased collagen contraction after collagen receptor (ß1-integrin) blockade (Figure 3Down). Furthermore, HA did not significantly alter cell numbers over the course of the contraction assay (HA 50%, 16.1±1.9 cells/mm2 versus collagen alone, 15.5±1.9 cells/mm2 at 18 hours, P=NS). However, HA did induce a significant change in cell shape and increased the pericellular accumulation of collagen fibrils (see below).



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  		Figure 2. Effect of increasing HA concentration on collagen gel contraction by smooth muscle cells at 18 hours. The effect was also seen with adventitial fibroblasts (not shown). HA caused a dose-dependent increase in gel contraction (decrease in gel diameter) compared with collagen alone (top). This was accounted for by CD44 adhesion, because pretreating cells with a blocking antibody specific for CD44 (1, 25, and 50 µg/mL) attenuated the affect of HA. Blocking CD44 (5 µg/mL) on cells seeded into collagen alone also inhibited contraction, likely by blocking adhesion to endogenously produced HA in the pericellular coat (middle). The CD44 affect was specific, because functional inhibition of RHAMM did not inhibit contraction of gels with or without HA (bottom). Each bar represents the mean±SD for triplicate gels in a representative experiment; *P<0.05; {dagger}P<0.01; {ddagger}P<0.001.



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  		Figure 3. Effects of HA on collagen gel contraction by smooth muscle cells in the presence or absence of ß1-integrin blockade. ß1 blockade (25 µg/mL anti-ß1 antibody) inhibited collagen contraction, but adding HA resulted in significant contraction in the presence of ß1 blockade. {dagger}P<0.01; {ddagger}P<0.001.

Enhanced collagen contraction was mediated by cellular binding to HA via CD44 receptors. Blocking CD44 with an antibody before seeding cells into gels completely inhibited the HA effect (Figure 2Up). A small but significant (P<0.05) inhibition of gel contraction was also observed after blocking CD44 on cells seeded into collagen alone (Figure 2Up). This likely represents inhibition of CD44 binding to endogenously produced HA present in the pericellular matrix coat. In contrast, ligation of RHAMM receptors by HA was not required for enhanced contraction, because pretreating cells with an antibody that inhibits RHAMM function10 14 had no effect on gel contraction (Figure 2Up). The effect of receptor blockade was similar for both smooth muscle cells and adventitial fibroblasts, and pretreating cells with nonimmune IgG1 showed no effect on contraction over the same range of concentrations.

Cell Shape Change and Collagen Reorganization
Although cell density and distribution were similar among gels, the addition of HA resulted in a significant change in cell shape. HA caused smooth muscle cells and adventitial fibroblasts to become more spread and to project long telopodia, as documented by {alpha}-actin immunofluorescence (Figure 4Down). The shape change was seen within 6 hours of polymerizing gels and was not inhibited by blocking CD44, ß1-integrins, or RHAMM. Along with an increase in cell spreading, HA also increased the accumulation of pericellular collagen fibrils. Away from individual cells, Sirius red–stained collagen fibrils were distributed homogeneously throughout gels with and without HA. However, in gels containing HA, collagen staining was intense in the pericellular space where Sirius red–labeled fibrils had accumulated, often tangential to the cell surface (Figure 4EDown). CD44 blockade inhibited the increase in pericellular collagen accumulation in gels containing HA (Figure 4FDown). Blocking ß1-integrins in gels of collagen alone inhibited localization of collagen at the cell surface, but in the presence of HA, collagen accumulation around cells was prominent even with ß1 blockade (data not shown). The effect was independent of MMP collagenase activity, because pretreating gels with batimastat had no effect on gel contraction or the increase in pericellular collagen in the presence of HA for either cell type.



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  		Figure 4. Effects of HA on cell shape and collagen reorganization. Gels are shown containing smooth muscle cells in collagen (A and D), collagen plus HA (B and E), and collagen plus HA with CD44-receptor blockade (C and F). Gels were fixed and embedded after 18 hours, and sections were stained with a fluorescent {alpha}-actin antibody to delineate the cytoskeleton and thus cell shape (A through C). Adjacent sections were stained with Sirius red and viewed under polarized light to identify collagen fibrils (D through F). Compared with cells in collagen alone (A), HA resulted in enhanced cell spreading and telopodia outgrowth (B and C). The change in shape was accompanied by an increased accumulation of collagen fibrils near the cell surface (E) when compared with cells in collagen alone (D). Pretreating cells with a blocking antibody to the CD44 receptor prevented HA-induced accumulation of collagen at the cell surface (F) but had no affect on the HA-induced change in cell shape (C). Original magnification x400, all panels.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Artery wall remodeling is one of the most important factors regulating lumen caliber after angioplasty.1 2 20 Smooth muscle cells and adventitial fibroblasts repopulate sites of injury,3 22 and as healing progresses, cells remodel new and preexisting extracellular matrix. As a result, focal changes in artery wall geometry occur that often culminate in wall shrinkage and restenosis.1 2 20 The extracellular environment strongly influences cell behavior, but how changes in matrix composition affect connective tissue shrinkage is poorly defined. Collagen and HA are both abundant in new matrix accumulating within the artery wall at sites of injury,23 24 and their expression overlaps temporally and spatially.3 In the present study, we have linked HA to enhanced collagen remodeling by smooth muscle cells and adventitial fibroblasts in vitro. If HA also promotes collagen contraction in vivo, the relative amount of HA produced at sites of injury may be critical in regulating wall shrinkage and restenosis. The significance of this possibility is underscored by the modest decrease in artery wall size required to greatly reduce lumen caliber in an artery thickened by plaque and neointima.2

We hypothesized that HA would inhibit collagen contraction by smooth muscle cells and adventitial fibroblasts on the basis of reports implicating HA in scarless fetal wound healing.5 25 26 27 Early fetal skin has a high HA content, and incisions heal by regeneration without fibrosis or scar.5 25 HA content falls as the fetus matures, and wounds then heal by scarring with granulation tissue, contraction, and fibrosis.11 Exogenous HA can shift the phenotype of late fetal wounds from scarring to regeneration, suggesting a transition linked mechanistically to loss of HA.26 We found that HA actually enhanced collagen contraction and reorganization by fibroblasts and smooth muscle cells cultured from mature primate aorta. Although opposite our hypothesis, these data seem consistent with effects of HA on wound healing in adults. Fibroblasts invade adult wounds and produce new matrix rich in HA as the scaffolding for granulation tissue formation and collagen accumulation.4 27 Treating adult wounds with exogenous HA does not prevent scarring but may reduce inflammation, increase reepithelialization, cellular ingrowth, and wound closure, and improve subsequent collagen remodeling.28 29

The mechanism by which HA enhances collagen reorganization in wounds has not been defined, and the literature is conflicting. HA has been reported to increase invasion of collagen gels by nonvascular fibroblasts and to inhibit collagen lattice contraction by human umbilical vein endothelial cells and fibroblasts, but results are conflicting, with other studies demonstrating no effects on lattice contraction by fibroblasts.30 31 32 33 34 35 Results seem to be dependent on the cell type, tissue, and species of origin.31 33 35 The few studies that have addressed the effects of HA on vascular smooth muscle cells have shown enhanced replication and migration in culture,10 36 37 and these effects were linked to CD44 and RHAMM receptor ligation, respectively.10 13 36 Although replication and migration are central to intimal hyperplasia, their role in constrictive arterial remodeling is unknown, and no previous reports have detailed the effects of HA on collagen reorganization by vascular smooth muscle cells or adventitial fibroblasts in vitro or in vivo. Herein we have shown that HA increases collagen gel contraction by aortic cells through interactions with CD44 but not with RHAMM. However, the mechanism underlying enhanced contraction was not an increase in cell number. Cell counts were similar for gels with and without HA at 24 hours, and the HA effect on contraction was seen as early as 4 to 6 hours of seeding gels, precluding a significant impact from altered replication. Rather, a CD44-dependent increase in pericellular collagen was observed, suggesting that cellular HA binding enhanced the recruitment of collagen fibrils to the cell surface.

The ß1-integrins are the primary cellular receptors for collagen, and, as previously described,17 a ß1-antagonist completely abolished contraction of collagen gels by vascular smooth muscle cells. We found an identical effect on adventitial fibroblasts. Adding HA, however, increased the extent of collagen contraction and allowed for significant contraction even after ß1 blockade. Both effects were CD44-dependent. Collagen can bind directly to HA,38 and binding is enhanced by matricellular proteins, such as fibronectin,38 present in serum required for contraction assays. Thus CD44-HA binding may provide an additional mechanism for vascular cells to interact (albeit indirectly) with collagen. Once bound to CD44, HA is taken up by cells7 9 39 and thus may chaperone collagen to the cell surface during the process of internalization. Given the significant length of HA chains, each molecule can link multiple collagen fibrils near to or distant from the cell surface and thus amplify forces generated through HA binding and internalization across the collagen lattice. CD44 can also bind collagen directly,40 41 and if HA significantly induced CD44 expression, a direct interaction may have contributed to HA-enhanced collagen reorganization.

Previous studies have also suggested that collagen gel contraction results from frictional forces caused by cell movement or spreading rather than cell shortening and contraction.42 Although we did not measure migration, we did use an anti-RHAMM antibody known to inhibit HA-induced migration of vascular smooth muscle cells in 2-dimensional cultures.10 36 Inhibiting RHAMM failed to alter gel contraction in the present study. This is in contrast to a recent report by Bagli et al,43 who blocked contraction of collagen gels (without exogenous HA) by rat bladder smooth muscle cells using a RHAMM-blocking peptide. The peptide adheres to RHAMM-binding sites on HA to prevent receptor ligation, whereas the antibody used in the present study inhibits RHAMM signaling but not binding to HA. However, we have compared effects of the blocking peptide to the anti-RHAMM antibody in our system with identical results (R.L. Geary, unpublished data, February 2000). The varied role of RHAMM in these two experiments may be attributable to differences in experimental design. We seeded cells into gels before polymerization. Bagli et al43 seeded cells onto the surface of gels after polymerization, which would have required cells to first invade the collagen lattice to effect contraction, and invasion may have been more RHAMM-dependent than the changes in cell shape observed in the present study. Other obvious differences include cell species (rat versus monkey) and tissue of cell origin (bladder versus artery wall).

HA is known to induce cell spreading in vitro,32 44 and we documented similar changes in cell shape in the present study. Surprising, however, was the apparent disconnect between enhanced contraction and altered cell shape in gels with HA, where blocking CD44 inhibited the former but not the latter. In contrast, Oliferenko et al45 recently reported directional outgrowth of lamellipodia from individual immortalized mouse epithelial cells adherent to plastic when HA was applied locally to the cell surface. The effect was mediated by CD44 activation of Rac1 and inhibited by blocking either the receptor or downstream kinase. The 3-dimensional format of the present study would not have provided a similar gradient of HA to invoke directional responses. Although inhibiting RHAMM or CD44 did not prevent the observed change in cell shape, we cannot exclude the participation of other potential HA receptors (eg, TSG-6 or intercellular adhesion molecule-139 ). Independent effects of HA on gel physical properties may also have contributed, because HA is known to increase the spacing between collagen fibers and gel stiffness.46 47 Increased stiffness, in particular, would be expected to alter cell polarization and spreading, but additional experiments will be necessary to define the mechanism underlying cell-shape change and whether it is necessary, if not sufficient, to effect HA-enhanced collagen reorganization.

Artery wall remodeling is achieved in part through turnover of new and existing matrix, and collagenolytic MMPs are induced within the artery wall after angioplasty. MMP activity has been implicated in constrictive remodeling, and the broad-spectrum MMP inhibitor batimastat has recently been shown to reduce the extent of wall shrinkage after peripheral angioplasty in pigs.20 A related MMP inhibitor has been shown to decrease collagen gel contraction by human dermal fibroblasts.48 To address the possibility that HA enhanced collagen reorganization by increasing expression or activation of MMPs, we treated cells with a range of batimastat concentrations known to block smooth muscle cell collagenolytic activity.49 In contrast to previous reports, MMP inhibition did not prevent or reduce collagen gel contraction48 and the effects of HA were unaltered. Thus, it is unlikely that increased collagen degradation contributed substantially to collagen reorganization by aortic smooth muscle cells and fibroblasts in the present study.

To our knowledge, no reports in the literature have specifically addressed the role of HA in artery wall remodeling in vivo. However, there are in vivo data supporting a role for both in the arterial response to injury. Deposition of HA3 6 13 and expression of CD4413 increase within the artery wall after angioplasty. Exogenous HA may blunt the response to angioplasty, because Ferns et al16 found that treating rabbits with HA after angioplasty reduced intimal thickening. This may have been an indirect effect, however, because artery wall inflammation was significantly reduced in treated animals at the site of injury. HA given systemically may be rapidly degraded to small fragments that can bind CD44 receptors on leukocytes to inhibit adhesion to tissue HA and recruitment into sites of injury.8 9 40 Whether small HA fragments also block the function of CD44 on smooth muscle cells and fibroblasts within the artery wall is not well defined, and Ferns et al16 did not measure changes in artery wall geometry. Varying HA chain length has resulted in differential responses of vascular cells in culture. Low molecular weight HA induced an angiogenic response from endothelial cells whereas high molecular weight HA was inhibitory,15 50 and small HA fragments inhibited smooth muscle cell movement whereas high molecular weight HA enhanced migration.36 Clustering of CD44 receptors by cytoskeletal reorganization can enhance intracellular signaling and could explain altered responses to small HA fragments not capable of bridging receptors.9 39 We used only high molecular weight HA (>1x106 kDa) in the present study, and effects of low molecular weight HA fragments on collagen gel contraction are as yet unknown.

In summary, HA promotes shrinkage of collagen gels by primate aortic smooth muscle cells and adventitial fibroblasts with associated changes in collagen reorganization and cell shape. Enhanced gel contraction and pericellular accumulation of collagen fibrils are mediated by CD44-receptor binding independent of ß1-integrin function. In contrast to previous studies using cells not of vascular origin, collagen lattice contraction did not require RHAMM receptor function or MMP activity, and neither accounted for the observed HA effects. In conclusion, the composition of the extracellular matrix may dramatically influence cell behavior and, thereby, tissue remodeling. Strategies to alter the balance between HA and collagen produced at sites of injury or to inhibit cellular binding to HA via CD44 should be explored as potential targets to inhibit constrictive artery wall remodeling and restenosis.


*    Acknowledgments
 
This work was supported by the National Institutes of Health (RO1HL57557 to R.L.G.) and by awards from the American Heart Association and the Association for Academic Surgery (to M.G.H.). R.L.G. is a Brook’s Scholar of the Wake Forest University School of Medicine. The authors wish to thank Michelle Gammons for preparing the manuscript.


*    Footnotes
 
Original received February 8, 2000; resubmission received September 21, 2000; revised resubmission received October 26, 2000; accepted October 31, 2000.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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