DEVELOPMENTAL

BIOLOGY

143,235-247

(1991)

Fibronectin, Hyaluronan, Increased Ductus

and a Hyaluronan Binding Protein Contribute Arteriosus Smooth Muscle Cell Migration

to

“Intimal cushions” which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous i?l vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a lo-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded DA or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 & 3.1%, vs 16.2 -t 2.3%>, P < 0.01). Addition of GRGDS peptides (0.5 mW) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of cndothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 & 3.15 J at a concentration of 1 m&ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:lOO dilution, 1 &ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a lo-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in uim (i 1991 Academic Prws. Inr

crease in hyaluronan and a 5-fold increase in heparan sulfate (HS) accumulation in the extracellular matrix of DA endothelial cells compared to that of Ao and PA cells. These differences were also present in cells from late gestation lambs although less marked. DA smooth muscle cells secrete Z-fold more fibronectin (FN) compared to Ao and PA cells and respond to endothelial cell conditioned medium by secreting 2-fold greater amounts of chondroitin sulfate (CS) (Boudreau and Rabinovitch, 1991). These DA endothelial and smooth muscle-specific changes in extracellular matrix production observed in vitro may promote the migration of the DA smooth muscle cells into the subendothelial space previously described iz ~ivo. For example, increases in hyaluronan have been found in several developmental systems involving tissue remodeling, including the formation of chick endocardial cushions (Markwald et al., 1977; Bernanke and Markwald, 1982; Bernanke and Orkin, 1984a,b), cornea (Toole et ul, 1984), and mouse neural

INTRODUCTION

In late gestation, the ductus arteriosus (DA) develops “intimal cushions,” the formation of which is initiated by the accumulation of subendothelial extracellular matrix material, forming a space into which medial smooth muscle cells migrate (Gittenberger de Groot et al, 1985; deReeder et al., 1988). In previous studies we have demonstrated that vascular cells harvested from the fetal lamb DA, aorta (Ao), and pulmonary artery (PA) maintain phenotypic properties during early passage in culture (Rabinovitch et al, 1988, 1989), which include distinct patterns of extracellular matrix production (Boudreau and Rabinovitch, 1991). At 100 days of gestation (term = 145 days), a timepoint which precedes the formation of intimal cushions, there was a lo-fold in’ To whom correspondence should be addressed at Division of Cardiovascular Research, The Hospital for Sick Children, 555 IJniversity Avenue, Toronto, Ontario, Canada M5G 1X8. 235

0012-1606/91 Copyright All rights

$3.00

C 1991 hy Academic Press, Inc. of reproduction in any form reserved.

236

DEVELOPMENTALBIOLOGY VOLUME143.1991

mesenchyme (Copp and Bernfield, 1988). The ability of hyaluronan to bind large amounts of water is believed to result in expansion of the tissue space allowing for unimpeded movement of cells into the adjacent areas (Toole et al., 1984). In addition, Turley and Torrance (1984) have described a cell surface-specific hyaluronan binding protein (HABP) which mediates cell movement in a hyaluronan-rich environment (Turley et al., 1985), and increased expression of this protein is found in association with actively motile cells (Turley and Torrance, 1984). Increased fibronectin has been described in cellular migration (Thierry et al., 1989), including development of cardiac cushions (Icardo and Manasek, 1984) and wound healing (Clark et al., 1982), and is believed to mediate the adhesion of cells to their extracellular environment (Hynes, 1982). As well, chondroitin sulfate has been implicated in increased motility of cells (Yamagata et al., 1989; Kinsella and Wight, 1986). Three-dimensional collagen gel matrices have been used extensively to study the migration of both vascular (Delvos et al., 1982; Wren et al., 1986) and nonvascular cells (Elsdale and Bard, 1972; Schor, 1980; Docherty et al., 1989; Robertson et al., 1989). As well, addition of various extracellular matrix components to these gels results in formation of matrices that more closely resemble those found in viva (Turley et al., 1985) and that provide a good model system to study the individual contribution of a particular matrix component to cell movement (Bernanke and Markwald, 1984). In this study we used three-dimensional collagen gels to investigate whether the previously described alterations in DA extracellular matrix components might be associated with increased DA smooth muscle migration. We compared DA with Ao smooth muscle cell migration and used antibodies directed against fibronectin as well as RGD peptides which block cellular binding to fibronectin. The effect of elevated chondroitin sulfate was investigated using endothelial cell conditioned medium to stimulate DA production of chondroitin sulfate. The contribution of a hyaluronan-rich subendothelium was explored using gels incorporating hyaluronan. A cell surface HABP was demonstrated using immunofluorescent staining, and the role of this protein was investigated by assessing migration in the presence of an antibody to HABP. Quantitation of HABP was assessed by immunoprecipitation of culture media and cell lysates following radiolabeling of cells with [35S]methionine. MATERIAL AND METHODS Cell Culture DA and Ao smooth muscle cells were harvested from fetal lamb vessels removed en bloc and quickly dissected. The lambs were delivered by cesarean section on Day 100 of a 145-day gestation period, as previously de-

scribed (Rabinovitch et al., 1988). Endothelial cells were harvested by scraping the luminal side of the vessel with a No. 11 scalpel blade (Ryan et ab, 1978) and smooth muscle cells were propagated by tissue explant (Ross, 1971). Endothelial and smooth muscle cells were positively identified by staining for Factor VIII and smooth muscle actin, respectively (Rabinovitch et ab, 1988). Smooth muscle cultures were maintained in Medium 199 (M199; GIBCO, Burlington, Ontario, Canada) containing 10% heat-inactivated FCS (GIBCO) and 1% antibiotic/antimycotic; cells were used at passage 2. The use of heat-inactivated serum would eliminate the possible effects of complement in the migration assays. Preparation

of Gels

Collagen gels were prepared according to the methods of Wren et al. (1986) and Elsdale and Bard (1972). Briefly, 4 ml of a 3.1 mg/ml solution of bovine dermal type I collagen (Vitrogen 100, Collagen Corp., CA), 0.04 ml of 0.142 M NaOH, 0.4 ml of 10X M199, and 1.76 ml of IX Ml99 (GIBCO) were mixed at 4°C for a final collagen concentration of 2 mg/ml. Aliquots of 2 ml were added to each 35-mm petri dish (Nunc, Denmark) and allowed to polymerize overnight at 37°C in a 95% sir/5% CO, incubator. Prior to use the gels were rinsed three times with Ml99 containing 10% FCS. In some experiments hyaluronan (bovine vitreous humor; Sigma, St. Louis, MO) was dissolved in Ml99 and added to the final gel solution at concentrations of 0.5-1.5 mg/ml. Harvest

of Smooth Muscle Cells and Seeding onto Gels

Confluent cells at passage 2 were collected by trypsinization and centrifugation as described by Schor (1980) and total cell number was determined by counting triplicate aliquots using a Coulter counter (Coulter Electronics, Hialeah, FL). Cell pellets were suspended in Ml99 plus 10% FCS and a l-ml aliquot containing 5 X lo4 cells was seeded onto the surface of each gel. Analysis

of Smooth Muscle Cell Migration

into Gels

Migration of cells into the gel matrix was assessed at 2,5, and 8 days following seeding by phase-contrast microscopy using a 10X objective (Nikon Diaphot inverted microscope; Nikon, Mississauga, Ontario, Canada). At each time point, in five randomly selected areas (0.92 x 1.30 mm) defined by the photographic grid on the lens, the number of cells on and below the gel surface was counted. The total number of cells within the gel was expressed as a percentage of the total cell number counted. To assess distance migrated, the number of cells at depths of 70-lOO, lOO-200, and greater than 200 pm were counted using the calibrated fine focus.

BOUDREAU,TIJRLEY,

Measurement of Attachment Eficiency

ANDRABINOVITCH

and Cell Nu,mber

Sixty minutes following seeding of cells onto the gels, attachment efficiencies were determined by counting the number of cells in the medium using a Coulter counter. The number of attached cells (number of cells seeded ~ number of cells in the medium) was expressed as a percentage of the number seeded. To assess the number of cells retained in the gels throughout the experimental period, i.e., 8 days following seeding, the gels were digested with 1.0 mg/ml collagenase type II (Sigma) for 3 hr at 37°C. Free cells were pelleted by centrifugation at 4°C for 10 min at 3008 and suspended in PBS containing 0.05% trypsin (GIBCO) and cell number was determined by counting aliquots in triplicate on a Coulter counter. Addition oj’ Conditioned Medium, RGD Peptides, and Ant ibodies Endothelial conditioned medium was added to smooth muscle cultures to stimulate synthesis of GAGS. Although HS and hyaluronan are stimulated to a similar degree in both DA and Ao, our previous studies have shown that in the presence of DA endothelial-conditioned medium, DA smooth muscle cel!s responded by secreting twofold more CS than corresponding Ao cells in the presence of Ao endothelial conditioned medium (Boudreau and Rabinovitch, 1991). The medium was collected following incubation with DA or Ao endothelial cells for 48 hr in Ml99 plus 10% FCS and was stored at -20°C until use, when 1 ml was added to the respective smooth muscle cells plated on each gel. To assess the contribution of fibronectin to cell migration, the stable peptide, GRGDS, or the inactive analogue, GRADSP (Telios Pharmaceuticals, San Diego, CA), was added to the culture medium at a final concentration of 0.5 rnJf. In separate experiments, polyclonal rabbit anti-human fibronectin antibodies (Chemicon, San Mateo, CA) were used at a dilution of 1:lOO. To block the effect of the hyaluronan binding protein, a polyclonal HABP antibody was used at a 1:lOO dilution (1 pg/ ml). This antibody is immunospecific for HABP and does not react with fibronectin, laminin, or collagens (Turley and Moore, 1984). A 1:lOO dilution of normal rabbit serum (Chemicon) was used as a control in all experiments with FN or HABP antibodies. In each of these experiments cells were allowed to adhere to the gels for 60 min prior to the addition of antibodies or peptides. Culture medium was not changed over the course of the g-day experiments.

DA and Ao smooth muscle cells at passage 2 were transferred 1:l to glass coverslips and allowed to grow

Sttcoofh Mtrsclc Miqrn tioic

237

for 3 days. Subconfluent cells were then fixed with freshly prepared 3.7% paraformaldehyde for 5 min and incubated with polyclonal antisera to a HABP diluted 1:40 in PBS containing 10% FCS. This protein was visualized using a 1:lOO dilution of goat anti-rabbit RITC (Miles). A similar dilution of normal rabbit serum was used as a control in each experiment. Following staining with antibodies photomicrographs were taken of 20 fields (x40 magnification) using a Nikon FX 35 camera. The slides were projected and the number of HABP-positive antigenic sites in each cell was counted by an observer blinded as to whether the cells were of DA or Ao origin. We also confirmed that anti-HABP reduced binding of hyaluronan to the cell surface. Cells were grown on cover slips and a 1:40 dilution of anti-HABP or normal rabbit serum was added to cells 16 hr prior to fixation in 4% paraformaldehyde. After blocking with 1.5% BSA, cells were then incubated for 1 hr with 1 pg/ml of biotinylated hyaluronan binding region of chondroitin sulfate proteoglycan, kindly supplied by Dr. Richard Margolis (Ripellino et a,l., 1985). After washing, cells were further incubated with a 1:50 dilution of FITC-avidin (Vector Labs) and photographed under identical conditions.

Flasks of confluent DA or Ao smooth muscle cells were incubated in 2 ml serum-free Ml99 for 24 hr. Conditioned medium was dialyzed against H,O, lyophilized, and reconstituted in 2~ SDS sample buffer. Following 10% SDS-PAGE, protein was transferred to nitrocellulose for 5 hr at 20 mA, according to the method of Towbin et ~1. (1979). The nitrocellulose was blocked for 1 hr with 3% BSA in 50 mMTris-buffered saline (pH 7.4) and incubated with a 1:50 dilution of polyclonal anti-HABP for an additional hour. Immunostaining was visualized using a 1:lOO dilution of rabbit PAP (Dako) reacted with DAB (Sigma). Irnrnu71OljreCir)itUtj071of HABP Subconfluent cultures of either DA or Ao smooth muscle cells were rinsed and labeled for 24 hr in the presence of 5 pCi/ml of [35S]methionine (NEN, Boston, MA) in Ml99 containing 10% FCS. Culture medium was removed and the protease inhibitors aprotinin, leupeptin, and pepstatin were added to a final concentration of 1 pg/ml each. Cells were lysed in 2 ml lysis buffer as previously described (Turley, 1989) and protease inhibitors were added. A loo-p1 aliquot was saved for determination of protein content using a Bio-Rad protein assay kit. Polyclonal antisera to HABP (20 pi/ml) was added to 1 ml of medium or cell lysate and allowed to shake overnight at 4°C. Protein A-Sepharose (50 ~1; lo%, w/v;

238

DEVELOPMENTAL TABLE

BIOLOGY

1

OFDA ANDAO SMOOTHMUSCLE

AWACHMENTEFFICIENCY

CELLS ON GELS Cell type

On type

DA Ao

I collagen

only

76.2 ? 8.4 85.2 k 3.2

Note. No differences were observed between expressed as percentage = 8 determinations.

+1 mg/ml

hyaluronic

acid

81.8 f 3.9 77.5 f 5.0

in the ability to attach to the gel substrates DA and Ao smooth muscle cells. Results are attachment 60 min following seeding for n

Pharmacia) was added for an additional 1.5 hr and the immune complexes were pelleted by centrifugation for 2 min at 7OOg.Pellets were washed five times in lysis buffer and resuspended in 50 ~1 SDS sample buffer. HABP was resolved by 10% SDS-PAGE under reducing conditions and gels were exposed to film (Kodak XOmat) for 1 week at -70°C. The bands in the gel corresponding to HABP were identified with the fluorograph, cut from the gel, solubilized in H,O,, and subjected to liquid scintillation spectrometry. Values were normalized to total protein content of each sample. Analysis of Data Unpaired Student’s t tests were used to compare migration values at each time point. For experiments with additional controls (e.g., normal rabbit serum), values were compared using ANOVA followed by Tukey’s tests to determine significant differences. Student’s t test was used to compare the number of HABP antigenic sites in DA vs Ao cells. RESULTS

Attachment Eficiencies DA or Ao smooth muscle cells did not differ in their ability to attach to the collagen substrata, as shown in Table 1, with approximately 80% of cells becoming attached within 60 min following seeding. As well, incorporation of hyaluronan into the matrices did not significantly affect the ability of either DA or Ao smooth muscle cells to attach to the gels. Cell Number No significant differences in cell number were noted when DA and Ao smooth muscle cells were compared over the S-day course of the migration experiments (Table 2). A slight increase in cell number in both DA and Ao cells was noted, however, in the presence of endothelial conditioned medium. The variability observed from experiment to experiment was similar to that which we had reported previously (Rabinovitch et al., 1988). The

VOLUME

143,199l

presence of antibodies to fibronectin or HABP did not significantly affect the number of either the DA or the Ao smooth muscle cells in the collagen gels as compared to normal rabbit serum control (1:lOO dilution in Ml99 + 10% FCS). As well, DA or Ao cell number did not differ significantly in the presence of GRGDS peptides when compared to control GRADSP (Table 2). Migration

of DA and Ao Smooth Muscle Cells

Following attachment to the collagen gels, the morphology of the DA smooth muscle cells seemed more elongated and spindle-shaped (Figs. lA, 1B) than that of the Ao cells, which were more stellate and flattened in appearance (Fig. 1C). With the addition of fibronectin antibodies, the DA cells adopted a more rounded, stellate appearance (Fig. 1D) resembling the Ao cells. No significant differences in DA and Ao smooth muscle cell migration through the collagen gels were observed on Days 2 and 5 following seeding (Fig. 2A). By Day 8, however, 37% more DA than Ao smooth muscle cells had invaded the gels (P < 0.01). In addition to greater numbers of DA compared to Ao cells migrating into the gels, the DA cells were found at deeper levels within the gels (Fig. 2B). Greater than 85% of all migrating Ao cells were found at depths between 70 and 100 pm, compared to 65% of DA cells. No Ao cells were at depths greater than 200 pm, while a significant proportion (10% ) of DA cells were found at this level. Role of Fibronectin in DA Smooth Muscle Migration To determine whether fibronectin was contributing to the increased DA cell migration, the latter was assessed in the presence of a 1:lOO dilution of polyclonal antisera to fibronectin or RGDS peptides (0.5 mM). As well, in the presence of the antibody (Fig. 3A) a significant reduction in DA mobility was observed on Day 5 which was persistent to Day 8 (P < O.Ol), resulting in rates of

TABLE

2

CELLNUMBERFOLLOWING~DAYSINCOLLAGENGELS DA

Condition Untreated +Endothelial conditioned medium Normal serum Anti-HABP Anti-FN GRADSP GRGDS Note. Cell values shown minations in * P < 0.05

73,111 93,143 102,951 97,273 118,719 101,387 80,360

Ao

k 13,136 k f f f f AI

5,849* 10,195 23,667 14,983 45,563 7,585

71,992 110,041 103,413 102,560 110,987 108,693 110,987

tr 11,213 k -+ f -t f +-

numbers were assessed on Day 8 of each experiment represent the means + SD for a minimum of three three separate experiments. as determined by unpaired Student’s t test.

17,998* 6,147 4,762 6,625 41,405 38,158 and deter-

BOUDREALJ,TURLEY,ANDRABINOVITCH

Srr/ooth MMh~

Mi{/rYc tiorr

FIG. 1. DA and Ao smooth muscle cells on collagen (2 my/ml) gels. (A) DA smooth muscle cells 2 days following seeding collagen gels. The cells exhibit a spindle-like elongated morphology and the majority of cells are visible on the surface of the indicates the outline of a cell which has migrated hrlow the surface of the gel. By focusing into the gel at a depth of 250 pm clearly into focus. (C) Ao cells, 2 days following seeding onto the surface of the gel, exhibit a flattened, stellate morpholo&T, antibodies against fibronectin (1:1(M) DA smooth muscle cells (Di also display a more flattened, stellate appearance (Bar

migration for DA cells similar to those observed previously for Ao. Although addition of fibronectin antiserum initially decreased migration of Ao smooth muscle cells at Day 2 (P < 0.05), values were similar to controls on Days 5 through 8 (Fig. 3B). Normal rabbit serum was used as a control in each experiment and did not significantly affect migration of Ao or DA smooth muscle cells. To determine whether fibronectin-dependent migration was mediated by the cellular recognition specific RGD sequence, an excess of GRGDS peptides (0.5 mM) was added to either DA or Ao smooth muscle

239

onto the surface of gels. The arrow in B (B), this cell comes In the presence of = 200 pm).

cells seeded onto collagen gels. Migration was significantly decreased in the DA cultures at Days 5 (P < 0.05) and 8 (P < 0.01) but had no effect on Ao cells (Figs. 4A, 4B). In the presence of the GRGDS peptides, migration of the DA cells was similar to that seen in the Ao. The inactive analogue GRADSP (0.5 mM) was used as a control in each experiment and did not alter migration in either DA or Ao cells. Although these experiments were conducted in the presence of FCS, which contains plasma FN, both DA and Ao smooth muscle cells would be exposed to similar amounts of this exogenous FN.

240

DEVELOPMENTALBIOLOGY A

VOLUME 143,199l

B 100

0 .

I DAY2

I

I

DAY 5

DAY 8

1

0 Ao

DA CELL

TYPE

FIG. 2. (A) Migration of DA (0) and Ao (0) smooth muscle cells seeded onto collagen (2 m&ml) gels over a period of 8 days. A significantly (**P < 0.01) greater percentage of DA cells were found to invade the gels by Day 8 in comparison to the Ao cells. Migration is expressed as a percentage of total cells and values represent the mean t SD from n = 8 determinations. (B) Proportion (5%) of cells that have migrated to distances of 70-100 pm (O), 100-200 km (El), or >200 pm (w) in the gels by Day 8 for each of eight experiments. The majority of Ao cells migrated at distances between 70 and 100 Wm. In the DA cells, a greater proportion of cells were found to have migrated distances of 100-200 or >200 Frn by Day 8, as compared to Ao

The different responses observed then would most likely be attributed to quantitative or qualitative differences in cellular FN. Role of Endothelial Conditioned Medium Endothelial conditioned medium did not significantly change DA or Ao smooth muscle cell migration at the time points studied (Table 3). Hyaluronan Migration

and HABP

in DA Smooth, Muscle Cell

Incorporation of hyaluronan into the collagen gels promoted migration of DA smooth muscle cells in a concentration-dependant manner (Fig. 5A). The greatest effect was seen at a concentration of 1.0 mg/ml in which migration was found to be significantly (P < 0.05) higher than that observed for DA cells on gels containing collagen only (30.7 & 3.9% vs 23.1 + 1.3%, Fig. 5B). In contrast to these findings, addition of 1 mg/ml hyaluronan to the gels did not increase migration of Ao smooth muscle cells (Fig. 5C). Antibodies directed against HABP (1:lOO dilution) were added to DA cells on gels containing 1 mg/ml hyaluronan and resulted in a significant (P < 0.01) decrease in migration (Fig. 6). The effect of blocking HABP on cell motility in the absence of a hyaluronan-rich environment was also assessed using DA or Ao cells plated on gels containing collagen only. In the presence of a 1:lOO dilution of antiHABP, migration of DA cells was significantly reduced by Day 8 (Fig. 7A). This reduced rate of migration was

similar to that observed in Ao cells which were unchanged in the presence of anti-HABP (Fig. 7B). A 1:lOO dilution of normal rabbit serum was used as a control in both experiments and had no effect on cell migration. Both DA and Ao smooth muscle cells stained positively for HABP but an increased intensity of granular staining was apparent in the lamellipodia of the DA cells (Figs. 8A, 8B). In addition, a greater (P < 0.001) number of positive antigenic sites were quantitated in the DA compared to the Ao cells (46.8 & 4.9 vs 26.5 * 2.2 sites per cell). The pattern of staining for HABP was distinct from that seen for fibronectin in both DA and Ao smooth muscle cells which appeared as fibrils over the cell body, but was noticeably absent from the lamellipodia (data not shown). Both DA and Ao cells showed extensive localization of hyaluronan over the cell surface (Figs. 9A, 9C). Preincubation of DA or Ao cells for 1 hr with antibodies to HABP resulted in a reduction in localization of hyaluronan to the cell surface (Figs. 9B, 9D). By Western blot analysis of culture medium from DA and Ao smooth muscle cells, antiserum to HABP recognized a major 66-kDa protein and other minor components (Fig. lOA). Using this polyclonal antiserum, two proteins of 66 and 55 kDa were specifically immunoprecipitated from radiolabeled culture media and cell lysates of the DA and Ao cells. A significantly greater amount (P < 0.05) of newly synthesized HABP was immunoprecipitated from DA conditioned medium, relative to the Ao (1223 +- 208 cpm/lOO pg protein vs 798 * 133 cpm/lOO Kg protein; Fig. 10B). Similar amounts of

BOUDREAU,

TURLEY,

AND RABINOVITCH

Smooth

Muscle

0 . DA\2

DA\5

241

Migratimz

1 DAM

I DAY5

I DAY0

DA;18

FIG. 3. Effect of antibodies against fibronectin on smooth muscle cell migration. (A) Addition of 1:lOO dilution of polyclonal antibody to fibronectin (---) significantly reduces migration of DA smooth muscle by Days 5 (*P < 0.05) and 8 (**P < 0.01) when compared to DA smooth muscle cells alone (-) or in the presence of 1:lOO dilution of normal (*P < 0.05) decrease in rabbit serum (. . e). (B) A small but significant migration of Ao smooth muscle cells was observed on Day 2 following seeding in the presence of 1:lOO dilution of fibronectin antibodies (---). No significant effect on Ao cell migration was noted on Days 5 or 8 when compared to Ao cells alone (-) or in the presence of a 1:lOO dilution of normal rabbit serum (. . .). Values represent the mean k SD for three to four determinations.

HABP were recovered from DA and Ao cell lysates (924 + 118 cpm/lOO gg protein vs 983 k 310 cpm/lOO pg protein; data not shown).

FIG. 4. Effect of RGD peptides on smooth muscle migration. Migration of DA smooth muscle cells on 2 mg/ml collagen significantly reduced on Days 5 (“P < 0.05) and 8 (**P < 0.01) presence of 0.5 mM GRDGS peptide (---) when compared with tion in the presence of 0.5 mM of the inactive analogue GRADSP (B) Migration of Ao smooth muscle cells on 2 mg/ml collagen unaffected in the presence of GRGDS (---) or GRADSP (-). represent the mean k SD for four experiments.

is in agreement with our earlier studies which did not show differences in the growth rates or attachment efficiencies of these cells on plastic substrata (Rabinovitch et ab, 1988). The increased DA smooth muscle motility does, however, appear to be related to cellular interac-

TABLE 3 EFFECT OF ENDOTHELIAL CONDITIONED ON SMOOTH MUSCLE MIGRATION

DISCUSSION

Our previous studies have shown that the endothelial cells from the DA produce a matrix rich in hyaluronan, and the corresponding smooth muscle cells secrete significantly greater amounts of fibronectin than cells from the adjacent vessel, the aorta (Boudreau and Rabinovitch, 1991). In this study we have demonstrated that the DA smooth muscle cells exhibit a greater ability to migrate into three-dimensional collagen gels than the smooth muscle cells from the Ao. As well, the DA cells migrate more deeply into the gels. The enhanced ability of the DA smooth muscle cells to migrate is not related to differences in either the degree of attachment to the gels or to the number of these two cell types. This

(A) gels is in the migra(-). gels is Values

Day DA +Endothelial medium Ao +Endothelial medium

2

Day

MEDIUM

5

Day

8

conditioned

9.7 t 1.6 10.7 k 4.2

17.0 +- 2.6 27.4 + 12.4

23.0 k 3.2 27.4 + 6.7

conditioned

8.5 2 1.4 10.2 * 0.7

16.1 k 3.9 19.6 +- 4.4

17.0 + 2.4 14.9 t 2.3

Nota No significant differences in the rates of migration in either DA or Ao smooth muscle cells were observed with the addition of endothelial conditioned medium. Results are expressed as percentage of cells within gels on Days 2,5, and 8 following seeding. Values represent mean 2 SD from a minimum of 4 determinations.

DEVELOPMENTAL

BIOLOGY

24-

-i/” 16 ’ , 0

I 1 mg Hyaluronic

40

1 2 acid

1

A-----DAY 2

DAY 5

DAY 6

DA;5

DA;6

30 1

1

- .

DA;12

’

’

FIG. 5. Incorporation of hyaluronan into collagen gels and the effect on smooth muscle cell migration in collagen (2 mg/ml) gels. (A) Concentration-dependent migration of DA smooth muscle cells (0) on hyaluronan (hyaluronic acid) (0.5-1.5 mg/ml) supplemented collagen gels after 5 days. Each value represents the mean + SD for four determinations. (B) By Day 8, migration of DA smooth muscle cells on 1 mg/ml hyaluronan (---) incorporated into collagen gels is significantly (*P < 0.05) greater than migration of DA smooth muscle cells seeded onto gels containing collagen only (-), 1~ = 8. (C) Migration of Ao smooth muscle cells is not affected by inclusion of hyaluronan (1 mg/ml) into gels (---) when compared to migration on gels containing collagen only (-). Values represent the mean f SD for at least four determinations.

tions with fibronectin and the distribution of an HABP. By immunogold electron micropscopy, we have previously observed a gradient of FN increasing toward the lumen of the DA (Boudreau and Rabinovitch, 1991). This gradient may serve to initiate directional migration of DA smooth muscle cells towards the subendothelium. Together with the finding that the DA smooth muscle cells selectively respond to a hyaluronan-rich environment, our results would suggest that the smooth muscle HABP would then permit interaction with HA

VOLUME

143,1%1

present in the subendothelium, leading to the increased presence of smooth muscle cells in the subendothelium observed in vivo. The baseline rates of migration in our studies are two times greater than those reported by Wren et al. (1986) for adult bovine aortic smooth muscle cells, but the difference is likely due to the fact that we used fetal cells, which have previously been described as being more motile in collagen gel assays than cells from older animals (Schor, 1980). In our previous studies we established that the addition of endothelial conditioned medium to DA smooth muscle cells stimulated a significant increase in their secretion of chondroitin sulfate (Boudreau and Rabinovitch, 1991). Although high concentrations of chondroitin sulfate in lamellipodia suggest that it has a role in reduced cell adhesion and locomotion (Yamagata et al., 1989), we found that the addition of endothelial conditioned medium had no significant effect on the rate of migration of DA smooth muscle cells. Chondroitin sulfate may, however, play a more important role in vivo as it has been shown to selectively bind hyaluronan (Hassell et ah, 1986; Turley and Roth, 1980) and thus may act to stabilize extracellular hyaluronan and facilitate its interaction with the HABP. We did note, however, that in the presence of endothelial conditioned medium, the numbers of both DA and Ao smooth muscle cells increased significantly. This effect is likely attributed to the presence of endothelial-derived growth factors such as FGF (Rifkin and Moscatelli, 1989), PDGF (Ross, 1986), or TGFP (Goodman and Majack, 1989) which have previously been demonstrated to stimulate growth of smooth muscle cells. Although we have also shown that DA endothelial cells produce a matrix enriched in heparan sulfate, it

- . DA;2

’

DA\

5

DA;

0

FIG. 6. The effect of blocking HABP on DA smooth muscle migration in hyaluronan-supplemented collagen gels. A 1:lOO dilution of antibodies against HABP (0) significantly (*P < 0.05, **P < 0.01) reduces migration of DA smooth muscle cells (-) on gels containing 1 mg/ml hyaluronan. A 1:lOO dilution of normal rabbit serum was used as a control (. . . ). Values represent the mean f SD for 4 determinations.

BOUDREAU,

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FIG. ‘7. The effect of blocking HABP on smooth muscle migration in gels containing collagen only. (A) Migration of DA smooth muscle cells into gels containing 2 m&ml collagen (-) is significantly (**, < 0.01) reduced by 8 days following seeding in the presence of 1:lOO dilution of antibodies against HABP (---) when compared to migration of DA smooth muscle cells without antibodies. A 1:lOO dilution of normal rabbit serum ( * . . ) was used as a control. (B) Migration of Ao smooth muscle cells on gels containing collagen only (-) was not affected in the presence of 1:lOO dilution of antibodies against HABP (---); a 1:lOO dilution of normal rabbit serum (. * *) was used as a control. Values represent the mean k SD for four determinations.

was not possible to test the effect of collagen gels containing this glycosaminoglycan on smooth muscle migration, as the presence of heparan has been shown to impair polymerization of collagen fibrils (Guidry and Grinnel, 1987). Once polymerized, however, the stability of the collagen gels does not appear to be affected by the presence of heparan, as no evidence of gel dissolution was noted in cultures where endothelial conditioned medium had been added to stimulate glycosaminoglycan synthesis. The reduction in DA smooth muscle motility in the presence of antibodies to fibronectin or an excess of RGDS peptides indicates that cell interactions with fibronectin are necessary for the augmented migration observed. Fibronectin-dependent migration requires a critical degree of cell adhesion and spreading to support locomotion, but falling short of strong focal contacts and excessive spreading that impede migration. The loss of the fusiform appearance of the DA cells when fibronectin is blocked suggests that it is contributing to the

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spindle-like morphology characteristic of actively motile cells (Docherty el al, 1989; Delvos et ah, 1982). Our previous reports have shown that the increase in fibronectin secreted by DA smooth muscle cells was not accompanied by increased accumulation of fibronectin in the matrix (Boudreau and Rabinovitch, 1991). A lack of fibronectin assembly in the matrix has been reported in actively motile fibroblasts and is attributed to a diffuse pattern of surface receptors for fibronectin (Roman et ah, 1989; Akiyama et ah, 1989). We are currently investigating the distribution of these receptors and their role in the enhanced migration observed in the DA smooth muscle cells. Although it has been suggested that hyaluronan can expand tissue spaces and alter physical restraints to movement (Toole et al., 1984), the finding that DA but not Ao smooth muscle cells respond to a hyaluronanrich environment indicates that specific cellular recognition of hyaluronan is required to promote movement. Our studies indicate that in DA smooth muscle cells, HABP appears to be prominent and concentrated in the leading edges and lamellipodia of these cells. A similar distribution of HABP in lamellipodia has been noted in highly motile fibroblasts (Turley and Torrance, 1984; Turley et ah, 1989). As well, immunoprecipitation revealed that DA cells have the capacity to synthesize greater amounts of HABP. Interestingly, this difference was reflected largely by HABP shed into the culture medium, as cell associated values of newly synthesized HABP were similar. It has been established that following cessation of migration in fibroblasts, HABP is subsequently shed into the culture medium (Turley et ah, 1989). As the immunoprecipitation studies of HABP were carried out in nonmigrating cells in culture flasks, newly synthesized HABP would likely be shed into the culture medium. Our findings demonstrate that blocking hyaluronan interaction with this protein inhibits hyaluronan-induced migration in DA cells in collagen gels, which is consistent with HABP acting as a cell recognition site for hyaluronan. HABP-hyaluronan interactions have previously been implicated in fibroblast locomotion (Turley et al., 1985) and antibodies against HABP have been demonstrated to impair locomotion in ras-transfected fibroblasts (Turley et al., 1989). Our studies have confirmed the ability of antibodies to HABP to reduce hyaluronan localization on DA cells. These findings of hyaluronan-HABP dependent migration in DA smooth muscle cells, along with our previous study showing production of a DA endothelial matrix rich in hyaluronan (Boudreau and Rabinovitch, 1991), indicate how the increased smooth migration observed in viva may likely occur. This HABP has been found to exist as a complex with a protein kinase, which is selectively activated by hyaluronan (Turley, 1989), and thus suggests a mechanism

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FIG. 10. (A) Western immunoblot of HABP recovered from culture media of DA or Ao smooth muscle cells. Proteins were separated under reducing conditions by 10% SDS-PAGE as described in text, stained with a 1:50 dilution of anti-HABP, and compared to 50 pg of purified HABP. Standard HABP appeared as a major band M, 66, with DA medium showing stronger staining than Ao. A number of smaller bands also appeared to cross-react with this antibody. (B) Autoradiograph of [%S]methionine-labeled DA and Ao smooth muscle cell media immunoprecipated with a 1:40 dilution of anti-HABP, showing greater amounts of HABP recovered from DA cells. Two bands of 66 and 55 kDa were specifically precipated with this antibody as indicated by the arrowheads.

by which hyaluronan binding to the cell may actively promote locomotion. In addition, studies by Turley et al. (1990) and Lacy and Underhill (1987) have indicated that cell surface hyaluronan binding proteins are linked to actin filaments of the cytoskeleton, also suggesting an active role in locomotion. The finding that migration

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of the DA but not Ao cells was inhibited by antibodies against HABP would argue against a generalized “paralysis” of the cytoskeleton arising from exposure to these antibodies. Also, blocking HABP reduces the level of migration of the DA cells to that seen in the Ao, suggesting that HABP is contributing to the enhanced migration of DA cells observed when plated on gels containing collagen only. As both DA and Ao smooth muscle cells produce similar amounts of hyaluronan in vitro (Boudreau and Rabinovitch, 1991) the differential response to blocking HABP is not related to differences in smooth muscle production of hyaluronan. On the basis of our findings, that by independently blocking fibronectin or HABP it is possible to reduce the degree of migration of DA cells to the level of the Ao, it seems unlikely that the increased ability of the DA cells to invade the collagen gels arises from the individual contributions of each of these factors. Rather, these results would suggest that fibronectin and HABP are acting in a cooperative manner to allow greater migration by the DA cells. As the elongated spindle appearance associated with migration appears dependent on fibronectin, and the HABP appears concentrated in cellular projections associated with this morphology, it may be speculated that the increased production of fibronectin by DA cells facilitates extension of lamellipodia allowing HABP to interact with extracellular hyaluronan. Although fibronectin has been shown to demonstrate a slight degree of binding to isolated HABP (Turley and Moore, 1984), it is not likely that this is occurring in our in vitro system, as fibronectin and HABP show very distinct patterns of distribution in these smooth muscle cells. Previous histochemical studies by deReeder et al. (1988) demonstrated the presence of large amounts of hyaluronan in the full-term ductus arteriosus of beagle pups, which was not observed in the aorta or in poodle dogs with a congenitally patent DA, indicating an important role for hyaluronan in the closure of the ductus. In our previous studies we have observed a lo-fold increase in accumulation of hyaluronan in the DA subendothelial matrix, and a 2-fold increase in fibronectin secretion by smooth muscle cells of the DA, compared to Ao. We have now shown that these features along with the associated increase in HABP likely contribute to the mechanism of DA smooth muscle cell migration observed in vivo.

FIG. 8. Immunofluorescent staining of HABP to HABP and visualized by RITC goat anti-rabbit cells (B) a higher density of antigenic sites was in lamellipodia and leading edges as indicated FIG. 9. Fluorescent staining for hyaluronan presence of 2 pg/ml of a biotinylated hyaluronan of polyclonal antisera to HABP greatly reduced

on Ao smooth muscle cells (A) on glass coverslips using a 1:40 dilution of polyclonal antibodies IgG. Staining was mostly seen over the body of the cell. (Bar = 28 pm). In DA smooth muscle observed within cells as indicated by the open arrow. Positive staining for HABP was prominent by the closed arrows. on DA (A) and Ao (C) smooth muscle cells fixed on glass coverslips. Cells were incubated in the binding fragment of proteoglycan, followed by avidin-FITC. Preincubation with 1:lOO dilution binding of hyaluronan in both DA (B) and Ao (D) smooth muscle cells. Magnification, ~1200.

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Supported by a grant from the Medical Research Council of Canada, MT 8546. M.R. is a Career Investigator of the Heart and Stroke Foundation of Ontario. E.T. is a recipient of a National Cancer Institute of Canada Senior Scientist Award, and is currently in the Department of Cell Biology at the University of Manitoba, Winnipeg, Manitoba.

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