Arherosclerosis, 94 (1992) 135- 146 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/%05.00 Printed and Published in Ireland

135

ATHERO 04818

Heparin

stimulates proteoglycan synthesis by vascular smooth muscle cells while supressing cellular proliferation

Parakat VijayagopalaTb, Henry P. Ciolinob, Bhandaru Radhakrishnamurthy”‘C Gerald S. Berensona Departments of ‘Medicine,

and

bAnatomy, and ‘Biochemistry, Louisiana State University School of Medicine, 1542 Tulane Avenue. New Orleans. LA (USA)

(Received 27 December 1991) (Revised, received 26 February 1992) (Accepted 3 March 1992)

Summary We studied the effect of heparin on proteoglycan synthesis by bovine aortic smooth muscle cells in culture. Confluent, growth-arrested cells were incubated with [35S]sulfate, [ 3H]glucosamine or [ 3]serine in the presence of O-600 &ml heparin. Metabolically labeled proteoglycans secreted into the culture medium and associated with the cell layer were analyzed. In cultures treated with heparin there was a dosedependent increase in [35S]sulfate incorporation into secreted proteoglycans which reached a maximum (35% above controls) at 100 kg/ml heparin. At higher concentrations of heparin, the stimulatory activity declined and finally disappeared. Radioactivity in cell-associated proteoglycans increased significantly (16% above controls) only in cultures treated with 100 pg/rnl heparin. Heparin also produced similar increases in the incorporation of [3H]glucosamine and [3H]serine into secreted and cell-associated proteoglycans. While chondroitin sulfate, dermatan sulfate and heparan sulfate were elevated in the media, only chondroitin sulfate and heparan sulfate were increased in the cell layer. Heparin did not alter the degradation of proteoglycans. Heparin, while inhibiting the proliferation of subconfluent smooth muscle cells, also stimulated to a greater extent the incorporation of [35S]sulfate into proteoglycans. Other glycosaminoglycans, such as heparan sulfate, dermatan sulfate, heparin hexasaccharide and Sulodexide caused a significant but lesser stimulation of proteoglycan synthesis, while chondroitin sulfates and hyaluronic acid had no effect. Gel filtration chromatography of proteoglycans and their constituent glycosaminoglycans from heparin-treated and untreated cultures showed no differences in their molecular size. The results indicate that heparin can stimulate proteoglycan synthesis by vascular smooth muscle cells irrespective of their state of proliferation. This might have implications in vessel wall repair and arterial wall lipid deposition.

Key words: Heparin; Smooth muscle cell; Proteoglycan Correspondence lo: Gerald S. Berenson, M.D., Department of Medicine, LSU Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA.

synthesis

136

Introduction

Materials and Methods

Proteoglycans are ubiquitous components of all tissues including the blood vessel wall. They consist of a protein core to which one or more glycosaminoglycan chains are linked covalently. In the vessel wall, proteoglycans have such diverse functions as providing structural support and influencing viscoelasticity, permeability and hemostasis [l-3]. Because of their complex-forming ability with plasma apo B-containing lipoproteins, proteoglycans may also facilitate lipid accumulation in the vessel wall and thus contribute to the pathogenesis of atherosclerosis [4,5]. Vascular smooth muscle cells in culture synthesize proteoglycans. The major proteoglycans synthesized by these cells have been characterized as containing chondroitin sulfate with varying amounts of dermatan sulfate and heparan sulfate [6,7]. Proteoglycan metabolism by smooth muscle cells can be modulated under a variety of conditions. Thus, Wight et al. [8] reported that smooth muscle cell proliferation in vitro induced by serum and platelet-derived growth factor is accompanied by increased production of proteoglycans. In addition, factors such as the age of the cells, state of confluency and culture conditions can modulate proteoglycan synthesis by smooth muscle cells [3]. Heparin, a highly charged glycosaminoglycan produced by mast cells, is a potent inhibitor of smooth muscle cell proliferation in vivo [9] and in vitro [lo]. Heparin also stimulates the synthesis of fibronectin and thrombospondin in smooth muscle cells [ll]. Recently, Tan et al. [12,13] observed that heparin in the presence of endothelial cell growth factor increased the production of proteoglycans by human iliac smooth muscle cells. However, there has been no systematic investigation of the effect of heparin alone on proteoglycan synthesis by vascular smooth muscle cells. Therefore, in the present study, we investigated the effect of heparin and other glycosaminoglycans on proteoglycan synthesis by confluent and proliferating smooth muscle cells from bovine aorta and characterized the proteoglycans. The results indicate that heparin stimulates proteoglycan production in smooth muscle cells irrespective of their state of proliferation.

Materials

Dulbecco’s modified Eagle’s medium, fetal bovine serum, gentamicin (10 pg/ml), amphoteritin B (250 pg/ml) and Dulbecco’s phosphatebuffered saline, were purchased from Gibco (Grand Island, NY). Plastic tissue culture vessels were from Coming (Corning, NY). Chondroitin ABC lyase (Proteus vulgaris), chondroitin AC II lyase (Atherobacter aurescens), guanidine hydrochloride, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, n-ethylmaleimide, e-aminocaproic acid, benzamidine hydrochloride, chondroitin 4-sulfate (bovine trachea), chondroitin 6-sulfate (Shark cartilage) and dermatan sulfate (pig skin) were from Sigma Chemical (St. Louis, MO). (35]Sulfate (43 Ci/mg .sulfate) and [3H]glucosamine (25 Ci/mol) were from ICN Radiochemicals (Irving, CA). L-[G-3H]serine (20 Ci/mmol) was obtained from American Radiochemicals (St. Louis, MO). Ready-Solve scintillation fluid was from Beckman. Sephadex G-50, Sepharose CL-2B, Sepharose CL-4B and Sepharose CL-6B were from Pharmacia LKB Biotechnology (Piscataway, NY). Heparin (beef lung) was from Upjohn (Kalamazoo, MI). Hyaluronic acid was from ICN Immunobiologicals (Lisle, IL). Sulodexide, a low molecular weight preparation of a mixture of heparin and dermatan sulfate, was a gift from Alpha Phamaceuticals (Bologna, Italy). Heparin hexasaccharide was a gift from Dr. Michael T. Reed. Heparan sulfate was prepared from beef lung. Cell culture

Smooth muscle cell cultures were established from bovine thoracic aorta [14]. Explants of the vessel media were minced and plated out in a 25-cm2 plastic tissue culture flask in 3 ml culture medium (Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum, 5 ml/l gentamicin and 2.5 ml/l amphotericin B). Cells began to grow out from the explants in 3-4 days and reached confluence in 2-3 weeks. The explants were then removed and subcultures were established by removing cells with 0.1% trypsin 0.05% EDTA for 3 min at 37’C. Cells were split

137

1:3 into 25 cm2 flasks. The medium was changed every 3 days. Radiolabeling of proteoglycans

For most of the experiments, smooth muscle cells between 5th and 10th passage were subcultured into 12- or 24-well plastic tissue culture plates and allowed to grow to confluence. The medium was then removed, the cultures were rinsed twice with Dulbecco’s phosphate-buffered saline and refed with Dulbecco’s modified Eagle’s medium containing 0.4% fetal bovine serum (low serum medium). Cell growth was arrested by incubating cells in this medium for 24-48 h at 37°C [lo]. The cells were then rinsed with saline and incubated in the low serum medium containing 20 &i/ml [35S]sulfate and O-600 pg./ml heparin (O-l 50 pg/rnl uranic acid) or 100 &ml heparan sulfate, hyaluronic acid, chondroitin 4- or 6-sulfate, dermatan sulfate, heparin hexasaccharide, or 62.5 pg/rnl Sulodexide at 37°C. Each glycosaminoglycan sample had approximately the same amount of uranic acid (25 &ml). The duration of incubation is indicated in the legends of individual experiments. In some experiments, cells were incubated with 100 &ml heparin (25 &ml uranic acid) in the presence of either 20 &i/ml [35S]sulfate and 10 Qml [3H]glucosamine or 20 &i/ml [3SS]sulfate and 20 &i/ml t3H]serine for 24 h. We also labeled proteoglycans in proliferating smooth muscle cells. Briefly, smooth muscle cells were plated at a density of 7000 cells/cm2. The cells were allowed to attach and grow for 24 h in 20% serum-containing medium. The cells were then growth-arrested by switching to the low serum medium for 48 h. The cultures were then returned to the 20% serum-containing medium in the presence or absence of 100 &ml heparin and incubated for 24-72 h at 37°C. The cultures were labeled with 20 &i/ml [35S]sulfate for the last 24 h of each incubation period. Separate culture wells were counted for cell number. Isolation of proteoglycans

At the end of each labeling period the culture medium was removed from each well and chilled on ice. The cell layer was washed with 1 ml of

Dulbecco’s phosphate-buffered saline and the wash was combined with the medium fraction. Solid guanidine hydrochloride was added to the combined medium and wash to make the solution 4 M together with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 mM EDTA, 50 mM benzamidine hydrochloride, 10 mM nethylmaleimide and 0.1 M e-aminocaproic acid). The cell layer in each well was extracted with 2.0 ml of 4 M guanidine hydrochloride, 0.05 M sodium acetate (pH 5.8) containing 0.1% Triton X-100 and the protease inhibitors mentioned above for 24 h at 4°C. Both the medium and the cell layer extracts were then stored at -20°C. The extraction procedure removed virtually all (> 99%) of the labeled proteoglycans from the cell cultures. Unincorporated radioactive precursors were removed from medium and cell layer extracts by chromatography on a Sephadex G-50 column (1 x 25 cm) equilibrated and eluted with 8 M urea, 0.15 M NaCl, 0.05 M sodium acetate (pH 6.0), containing 0.5% (w/v) Triton X-100 and protease inhibitors. In the case of cells incubated with [ 35S]sulfate alone, the excluded material was pooled, an aliquot was dialyzed exhaustively against 0.15 M NaCl, 0.05 M Na2S04 containing protease inhibitors and the radioactivity incorporated into proteoglycans was measured by liquid scintillation spectrometry on a Beckman LS 3801 counter. (Preliminary studies indicated that almost all of the incorporated [35S]-label was in proteoglycans and therefore the 35Sradioactivity in the excluded fraction from the Sephadex G-50 column represented entirely proteoglycans.) Proteoglycans were purified from the remainder by DEAESephacel chromatography (see below). For cells incubated with [3SS]sulfate and [ 3H]glucosamine, the excluded fractions from Sephadex G-50 chromatography were applied to a column of DEAE-Sephacel (bed volume 8 ml) equilibrated in 8 M urea, 0.15 M NaCl, 0.05 M sodium acetate (pH 6.0), containing 0.5% Triton X-100 and labeled glycoproteins were eluted with 20 ml of the above buffer at a flow rate of 5 ml/h. Labeled proteoglycans were then eluted with a continuous NaCl gradient (from 0.15 to 1.O M) in the same solvent. The proteoglycan fractions were

138

pooled, dialyzed against 0.15 M NaCl (pH 7.0), containing protease inhibitors, and an aliquot was counted for both [35S]sulfate and [3H]glucosamine radioactivity. For cells incubated with [3sS]sulfate and [3H]serine an aliquot of the excluded material from Sephadex G-50 (medium and cell layer extracts) was dialyzed exhaustively against 0.15 M NaCl, 0.05 M Na2S04 and 0.001 M serine, containing protease inhibitors and assayed for radioactivity incorporated into non-dialyzable protein. The radiolabeled proteoglycans were then purified from the remainder of the dialysate by DEAE-Sephacel chromatography as above. Carrier chondroitin sulfate (500 pg) was added to the purified fractions and proteoglycans were precipitated in 1% 1-hexadecyl pyridinium chloride (CPC) at 25°C. The proteoglycans were then reisolated from their CPC complexes [15], dissolved in phosphate buffered saline and analyzed for radioactivity. Effect of heparin on total protein content and cell number Confluent smooth muscle cells in 24-well culture plates were incubated for 24-48 h in low serum medium. The medium was removed and the cells were mcubated in 0.5 ml of the same medium in the presence or absence of 100 pg/ml heparin for 24 h. At the end of the incubation period the medium was removed and the cells were rinsed twice with Dulbecco’s phosphate-buffered saline. Representative wells were assayed for protein [ 161; or trypsinized (as described previously) and counted by hemocytometry. Pulse-chase experiment The effect of heparin on proteoglycan turnover in smooth muscle cell cultures was investigated by pulse-chase experiments. Confluent, growtharrested smooth muscle cells in 6-well culture dishes (650 000 cells/well) were incubated with 20 &i/ml [3sS]sulfate for 20 h at 37°C in the presence or absence of 100 &ml heparin. After removing the medium, we washed the cell layer 10 times with Dulbecco’s phosphate-buffered saline containing 0.5 mM Na2S04. Separate wells were then chased for different times in isotope-free medium with or without heparin. After each chase

period proteoglycans were extracted from the cell layer. Labeled proteoglycans released in the media and remaining in the cell layer as well as free [ 3SS]sulfate generated by intracellular degradation of labeled proteoglycans were quantified by Sephadex G-50 chromatography. Characterization of proteoglycans Confluent, growth arrested smooth muscle cells in 25 cm2 culture flasks were incubated in low serum medium containing 20 &i/ml [3sS]sulfate in the presence or absence of 100 &ml heparin for 24 h at 37°C. Labeled proteoglycans in the medium and cell layer were extracted as described above and purified by DEAE-Sephacel chromatography. The proteoglycan peaks were separately pooled and rechromatographed on a second DEAE-Sephacel column. Portions of proteoglycan fractions from the second DEAE-Sephacel chromatography were /3eliminated with alkaline borohydride [17]. The types of glycosaminoglycans present were determined by susceptibility to nitrous acid [ 181, chondroitin AC11 lyase or chrondroitin ABC lyase [ 191. The relative portion of each glycosaminoglycan species was determined by quantifying the amount of radiolabeled material resistant to or degraded by each treatment. The remainder of proteoglycans was chromatographed on a Sepharose CL4B column (0.9 x 100 cm) in 4 M guanidine hydrochloride, 0.05 M sodium acetate (pH 6.0) containing 0.2% Triton X-100. Fractions (1 ml) were collected at a flow rate of 9.6 ml/h and aliquots were counted for radioactivity. Some of the proteoglycans from the Sepharose CL4B were eluted on a Sepharose CL-2B column (0.9 x 100 cm) using the above solution and fractions were counted for radioactivity. The molecular size distribution of the glycosaminoglycan side chains of proteoglycans from Sepharose: CL4B chromatography was examined. Glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride and chromatographed on an analytical CL-6B column (0.8 x 60 cm) in 0.2 M NaCl, 0.05 M Tris (pH 7.4) containing 0.2% Triton X-100. Fractions (0.5 ml) were collected at a flow rate of 10 ml/h and analyzed for radioactivity. The

139

elution positions of the glycosaminoglycans were compared to those of standards to determine their molecular weights [20]. Results Effect of heparin on [35SJsulfate and [“H]glucosamine incorporation into proteoglycans by smooth muscle cells

Figure 1 shows the effect of increasing concentrations of heparin on t3’S] sulfate incorporation into proteoglycans by confluent smooth muscle cells. There was a dose-dependent increase in [ “Slsulfate incorporation into secreted proteoglycans. Maximal stimulation of sulfate incorporation (35% above controls, P = 0.001) occurred in cultures exposed to 100 &ml heparin. Higher concentrations of heparin produced lesser but significant (P < 0.05) stimulation up to 400 pg/ml; at 600 &ml heparin there was no difference between control and experimental cultures. Incorporation of label into cell-associated proteoglycans was stimulated significantly only in cultures exposed to 100 &ml heparin (16% above controls, data not shown). In the above studies, we investigated the

possibility that heparin, especially at high concentrations (600 &ml), could precipitate the apo Blipoproteins in the culture medium, thereby making them inaccessible to the cells. We added heparin (1 mg/ml) to the low serum medium and incubated the solution at 37°C for 24 h. The solution was centrifuged at 15 000 rev./min to separate any precipitated proteins. There was no precipitation of proteins under these conditions. Earlier we have reported that irrespective of the amount of heparin added to serum, the apo B-lipoproteins were not precipitated below a Ca2+ concentration of 25 mM [21]. The Ca2+ concentration of our culture medium was only 1.8 mM. Time-course studies were performed in which growth-arrested cultures were incubated with [35S]sulfate in the presence of 100 &ml heparin for 6-48 h. As shown in Fig. 2A, incorporation of label into secreted proteoglycans increased almost linearly up to 48 h in both control and heparintreated cultures. However, at all time periods, the incorporation was significantly higher in heparintreated cultures as compared to controls. There was already a significant increase (11% above controls, P < 0.05) in [35S]sulfate incorporation in heparin-treated cultures after only 6 h incubation.

14000

2 ._

‘0

12000

kF

VI 10000 = O Q) ? v 8000 -m al 0 G 7 ~ 6000 E 3 v-l ,” 4000 W-IV

0

25

50 licparin

100

200

400

600

(jrg/ml)

Fig. 1. Incorporation of [3sS]sulfate into secreted proteoglycans by smooth muscle cells in response to heparin. Confluent, growtharrested smooth muscle cells were incubated with [3sS]sulfate for 24 h in the presence of O-606 &ml heparin. Labeled proteoglycans in the culture medium were isolated and quantified. Bars represent the mean stimulation by heparin in 9 cultures t SE. Heparin at each concentration produced a significant increase in [35S]sulfate incorporation over control (P C 6.05) except at 666 &ml.

140

lock 160-

go-

160-

60-

140-

TO-

B

Cell Lcl)nJr

504060-

F

30: 20 lo0

I I I1 I I 6 12 16 24 30 36

I I 42 46

0

Time (h)

I I I I I I 6 12 16 24 30 36

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Time (h)

Fig. 2. Effect of heparin on the incorporation of [35S]sulfate into proteoglycans as a function of time. Confluent, growth-arrested smooth muscle cells were incubated for 6-48 h with [35S]sulfate in the presence (O-O) or absence (0-O) of 100 &nl heparin. At the end of the indicated time intervals labeled proteoglycans in the culture medium (A) and cell layer (B) were isolated and quantified. Data represent the mean of 6 cultures f SE.

The stimulation of label incorporation was 35-40% above controls when cultures were exposed to heparin for 12-48 h. Incorporation of [ 3sS]sulfate into cell-associated proteoglycans was significantly higher (1 l-16% above controls) in heparin-treated cultures after 12 h incubation (Fig. 2B). Heparin (100 pg/ml) also stimulated [3H]glucosamine incorporation into secreted proteoglycans by 35% above control (control (dpm x 10m3 i S.E. per mg protein), 66.7 f 4; heparin treated, 90.5 * 4.2). [3’S]Sulfate incorporation in this double-label experiment was stimulated 38% above control (control, 130.6 f 3.8; heparin treated, 181 f 4.2 (dpm x lo-’ f S.E. per mg

protein)). Both [3H]glucosamine and [35S]sulfate incorporation into cell-associated proteoglycans increased 15% above control in cultures exposed to heparin (data not shown). The 31W35Sratios of proteoglycans from control and heparin treated cultures did not change. These results thus confirmed the stimulatory effect of heparin on proteoglycan production by smooth muscle cells. Effect of heparin on [3H]serine incorporation into total proteins and proteoglycans

Table

1 shows

the

effect of heparin on into non-dialyzable proteins. Heparin did not cause an increase in newly synthesized total proteins. In contrast, as com[ ‘Hlserine incorporation

TABLE 1 EFFECT OF HEPARIN ON THE INCORPORATION SMOOTH MUSCLE CELLS

OF [3H]SERINE INTO PROTEINS AND PROTEOGLYCANS

Numbers are dpm [3H]serine x 10b6/mg cell protein f SD. of triplicate cultures. Proteins

Control Heparin treated

Proteoglycan

Cell layer

Medium

Cell layer

Medium

6.3 + 0.42 6.1 f 0.50

7.8 f 0.62 8.1 f 0.69

0.34 f 0.05 0.42 f 0.10

1.8 f 0.2 2.6 zt 0.3

BY

** +* IIr-l-J 141

pared to control, heparin produced a 44% increase in the incorporation of [3H]serine into secreted proteoglycans. The corresponding increase for cell layer proteoglycans was 23%. In this experiment, heparin stimulated [ “Slsulfate incorporation into secreted and cell associate proteoglycans 38% and 16%, respectively, above control. However, heparin did not significantly alter the total cellular protein content or cell number of confluent smooth muscle cell cultures (data not shown).

*

I

Pulse-chase studies

Heo

To determine the effect of heparin on the degradation and secretion of proteoglycans, we performed a pulse-chase experiment. The results are shown in Fig. 3. The total [35S]labeled proteoglycans (cell layer + medium) in the control and heparin treated cultures decreased at about the same rate with time. At the end of the 24-h chase, control cultures and heparin treated cultures contained 29% and 33%, respectively, of the labeled proteoglycans originally present at time zero. The rate of degradation of labeled proteoglycans to free [35S]sulfate was also similar in both cultures. Thus, after the 24-h chase almost

h

HS

I

HA

I

I

I

I

c-4sos c-6s Hex

Sul

Giycosaminoglycan

*Significant

~(0.05

Fig. 4. Effect of different glycosaminoglycans on [%]suifate incorporation into secreted proteoglycans by smooth muscle cells. Confluent, growth-arrested cultures were incubated with [35S]sulfate for 24 h in the presence or absence of various glycosaminoglycans. Labeled proteoglycans secreted into the culture medium were isolated and quantified. Data represent mean of 6 cultures. Hep, heparin; HS, heparan sulfate; HA, hyaluronic acid; C-4S or C-6$ chondroitin 4- or 6-sulfate; DS, Dermatan sulfate; Hex, heparin hexasaccharide; Sul, Sulodexide.

the same percent of [35S]proteoglycans present at time zero was degraded in both cultures (control, 61%; heparin treated, 64%). 0 3

Celltkdum PG 0 Control ?? tkpoin

Free I%Sulfate 0 Control ?? Heparin

06 x

HOUS Fig. 3. Pulse-chase analysis. Confluent, growth-arrested smooth muscle cells were incubated with [35S]sulfate for 20 h in the presence or absence of 100 &ml heparin, washed extensively and chased for up to 24 h. At the indicated times the amounts of remaining labeled proteoglycans (cell layer + medium) and free [35S]sulfate generated by proteoglycan degradation were measured. Data represent the mean of 4 cultures.

Effect of other glycosaminoglycans on [:“s]surfate incorporation by smooth muscle cells

We compared the effect of other glycosaminoglycans with that of heparin on the incorporation of [35S]sulfate into secreted proteoglycans by smooth muscle cells. The results are shown in Fig. 4. As expected, heparin produced the highest stimulation. Heparan sulfate and Sulodexide stimulated [ 35S]sulfate incorporation by greater than 20% above control. Dermatan sulfate and heparin hexasaccharide stimulated incorporation by a lesser but significant (P < 0.05) level. In contrast, chondroitin 4- and 6-sulfates and hyaluronic acid did not increase [35S] incorporation significantly. Effect of heparin on proliferating smooth muscle cells

Heparin is a potent inhibitor of smooth muscle cell proliferation. Studies were conducted to deter-

142

mine whether inhibition of smooth muscle cell proliferation by heparin would influence the cells’ ability to synthesize proteoglycans. Smooth muscle cells were cultured at subconfluent densities, growth arrested and then returned to a growth state by the addition of 20% serum-containing medium. Cultures treated with 100 &ml of heparin had a significantly slower rate of proliferation, as compared to non-treated controls (Fig. 5; P < 0.02 to c 0.001). However, at this concentration, heparin significantly increased [“Slsulfate incorporation into medium proteoglycans on a per cell basis (45-60% above controls). Cell-associated incorporation also increased by 35-58% above controls. Characterization of proteoglycans [ 35S]-Labeled proteoglycans excluded from the

Sepahdex G-50 column were fractionated by DEAE-Sephacel chromatography (Fig. 6). The elution profiles were similar for the medium and cell layer fractions from control and heparin treated cultures. For each sample two [35S] peaks (indicated by 1 and 2) eluted from the column at NaCl concentrations of 0.48 M and 0.54 M, respectively, indicating that proteoglycans syn-

10 - A

thesized in the presence of heparin had similar charge densities to proteoglycans from control cultures. Proteoglycans in peak 1 and peak 2 fractions from DEAE-Sephacel chromatography were pooled separately and rechromatographed on a second DEAE-Sephacel column. Almost all of the [35S] radioactivity in peak 1 proteolgycans was susceptible to nitrous acid degradation. This indicated that these were heparan sulfate proPeak 2 contained chondroitin teoglycans. sulfate-dermatan sulfate proteoglycans as evidenced by their susceptibility to chondroitin ABC lyase digestion. Figure 7 shows the effect of heparin on glycosaminoglycan composition of proteoglycans in cell layer and medium. Compared to control, heparin treatment of cultures increased heparan sulfate (1.7-fold) and chondroitin sulfate (1.7-fold) in the cell layer. Dermatan sulfate content was not affected. In the medium, however, all three glycosaminoglycans were increased by heparin (heparan sulfate, 1.3-fold; dermatan sulfate, 2.2-fold; chondroitin sulfate, 1.7-fold). Total (cell layer + medium) chondroitin sulfate was increased the highest by heparin treatment (85% above control).

Cell Number

M

0

24

Time (h)

40

Time of Incubation

72

(h)

Fig. 5. Effect of heparin on cell proliferation and proteoglycan synthesis by proliferating smooth muscle cells. Smooth muscle cells were growth arrested at subconfluent densities and then released from growth arrest by adding 20% fetal bovine serum-containing medium. The cells were incubated with [3sS]sulfate in the presence or absence of 100 &ml heparin. Cell number (A) and [35S]sulfate incorporation (B) were assessed on separate cultures. Data represent mean of 6 wells for cell number and 4 wells for [35S]sulfate incorporation. Hatched bars, secreted proteoglycans; open bars, cell-associated proteoglycans.

143

"1 Medh?l

j _h

.I

-

Control t!qmin

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5._

x

Em

A

0

-3

w

0

10

20

30

;..:...,:

6

..,....._.. .’

‘,,. ..,,......... 8

‘....

.‘..

...

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40

FractionNurba Fig. 6. DEAE-Sephacel chromatography of [%]proteoglycans for control and heparin treated cultures. Confluent growth arrested smooth muscle cells were labeled with [35S]sulfate in the presence or absence of 100 pglml heparin for 24 h. Proteoglycans from cell layer and medium isolated by Sephadex G-50 chromatography were fractionated on DEAE-Sephacel. The proteoglycan Peaks 1 and 2 were pooled as indicated for further analysis.

Fig. 8. Sepharose CL-4B chromatography of chondroitin sulfate-dennatan sulfate proteoglycan. [3SS]-LabeIed chondroitin sulfate-dermatan sulfate proteoglycan isolated on DEAE-Sephaoel from the medium and cell layer of heparin treated and control cultures was subjected to Sepharose CL-4B chromatography. Fractions (1 ml) were collected at a flow rate of 9.6 ml/h and counted for radioactivity. Void volume (V,) and total volume (Vt) were determined with E. co/i and [35S]sulfate, respectively.

The proteoglycans from the DEAE-Sephacel chromatography were analyzed by Sepharose CL4B chromatography. Peak 1 from the medium and cell layer of control and heparin treated cultures eluted in the inclusive volume of the column as a single peak with identical profiles (data not shown). In contrast, peak 2 from the medium and cell layer was resolved into two peaks (Fig. 8, peaks A and B), one containing large pro-

Sdf ate

Sulfate

Sulfate

Fig. 7. [3SS]Glycosaminoglycans in the cell layer and medium of control and heparin treated cultures. Proteoglycan Peaks 1 and 2 from DEAE-Sephacel chromatography were separately rechromatographed on a second DEAE-Sephacel column and [3sS]glycosaminoglycans were isolated following treatment with alkaline borohydride. The glycosaminoglycans were then digested with chondroitin ABC lyase, chrondroitin AC11 lyase and nitrous acid to quantitate chondroitin sulfates, dermatan sulfate and heparan sulfate, respectively. Data represents the mean f SE. of triplicate analyses.

144

teoglycans (peak A) and the other containing smaller proteoglycans (peak B). The elution profiles were once again similar for control and heparin treated cells. Analytical Sepharose CL-2B chromatography of the peak A proteoglycan from the medium and cell layer of both cultures gave a single peak (K,,, = 0.31, data not shown). The smaller proteoglycan (peak B) from the medium and cell layer of control and heparin treated cultures also eluted as a single peak from the CL-2B column (K,, = 0.60; data not shown). These studies thus indicated that treatment of smooth muscle cells with heparin did not alter the size of the proteoglycans. Portions of the heparan sulfate proteoglycans and chondroitin sulfate-dermatan sulfate proteoglycans from CL=4B chromatography were treated with alkaline borohydride. The relative hydrodynamic size of the released glycosaminoglycan chains was then determined by analytical Sepharose CLdB chromatography. The heparan sulfate chains from the cell layer and medium of control and heparin treated cultures had the same average molecular weight (36 000). Similarly, the molecular weights of the two chondroitin sulfatedermatan sulfate chains from the cell layer and medium of both cultures were 40 000 and 36 000, respectively. Thus the average molecular weights of glycosaminoglycans from individual proteoglycans of control and heparin treated cultures were identical. Discussion The results presented in this report demonstrate that heparin, a highly charged glycosaminoglycan, stimulates proteoglycan synthesis by vascular smooth muscle cells. This is due to the direct effect of heparin on the cells and not the result of alterations of the culture media induced by the polysaccharide. Heparin significantly increased the production of both secreted and cell-associated proteoglycans, although the extent of increase was greater for secreted proteoglycans. While there was a stimulation in all proteoglycans released into the media, the increase was confined to heparan sulfate and chondroitin sulfate for the cellassociated proteoglycans. Cells exposed to heparin at concentrations up to 100 pg/ml responded with

a dose-dependent increase in proteoglycan production. At concentrations above this heparin exhibited a gradual loss of its stimulatory activity. The polysaccharide at these higher concentrations was not toxic to the cells. Also heparin does not produce growth inhibition of confluent smooth muscle cells [22]. Therefore the reason for the biphasic effect of the polysaccharide on proteoglycan synthesis by smooth muscle cells is not understood at present. Our observation that heparin stimulates proteoglycan production by confluent bovine aortic smooth muscle cells is in conflict with the findings of Nader et al. in rabbit aortic smooth muscle cells [23]. These investigators observed no stimulation of proteoglycan synthesis by heparin. The discrepancy may be due to the species difference. Alternatively, this could be due to the different experimental conditions used in the two studies. For example, we used confluent smooth muscle cells which were growth arrested and metabolic labeling was performed in medium containing 0.4% serum. Nader et al., on the other hand, used post-confluent cells which were labeled in medium containing loo/o serum. Heparin stimulated the incorporation of both radioactive-sulfate and glucosamine into proteoglycans in smooth muscle cells. In addition, the polysaccharide also produced a specific stimulation of [ ‘Hlserine incorporation into proteoglycan core protein without causing a general stimulation of protein synthesis. Pulse-chase studies demonstrated that heparin did not inhibit proteoglycan turnover. Thus, taken together, these results strongly suggest that heparin modulates the incorporation of labeled precursors into proteoglycans by stimulating their de novo synthesis. It is likely that heparin regulates synthesis at the level of translation or post-translational processing of proteoglycans. This is supported by a recent study in which a combination of heparin and endothelial cell growth factor depressed the mRNA levels of decorin proteoglycan in human vascular smooth muscle cells while causing an increase in its synthesis [13]. The action of heparin on smooth muscle cell proteoglycan production is not specific to this glycosaminoglycan. Other glycosaminoglycans such as heparan sulfate and dermatan sulfate also

14.5

produced a significant but lesser increase in proteoglycan synthesis by the cells (Fig. 4). Heparan sulfate was much more effective than dermatan sulfate. This could be due to the close structural similarities between heparan sulfate and heparin. Another structural feature common to the three glycosaminoglycans is their content of iduronic acid. The presence of iduronic acid in the glycosaminoglycan might be critical for its stimulatory effect on proteoglycan synthesis. That chondroitin 4- and 6-sulfates and hyaluronic acid, which do not contain iduronic acid, also did not stimulate proteoglycan synthesis supports this conclusion. A major finding in the present study is that heparin stimulates proteoglycan synthesis by smooth muscle cells even when it inhibits cellular proliferation (Fig. 5). This stimulation was even greater than that seen in confluent cultures. Thus, it appears that heparin enhances proteoglycan synthesis by smooth muscle cells irrespective of their proliferative state. This would imply that the polysaccharide’s abilities to inhibit smooth muscle cell proliferation and stimulate protoglycan synthesis are properties independent of each other. Heparin stimulates the synthesis of proteoglycans without affecting their molecular size or that of their constituent glycosaminoglycan chains. Also, proteoglycans produced by cells exposed to heparin had similar charge densities to those synthesized by control cells. This indicates that heparin did not cause oversulfation of proteoglycans in smooth muscle cells. This is contrary to the higher degree of sulfation of heparan sulfate proteoglycan reported by Nader et al. [23] in rabbit aorta endothelial cells exposed to heparin. Vascular smooth muscle cell proliferation occurs following vascular injury as well as vascular surgery. Smooth muscle cell proliferation is frequently associated with increased proteoglycan production [3]. Heparin is widely used to inhibit smooth muscle cell hyperplasia. However, in vivo, the polysaccharide stimulates the synthesis of heparan sulfate and chondroitin sulfate in rat carotid artery following balloon injury [24]. In vitro, as reported in our study, heparin is a potent stimulator of chondroitin sulfate proteoglycan synthesis by vascular smooth muscle cells even while it suppresses cellular proliferation. Taken

together, these observations might have physiological significance. The occurrence of heparin in aortic tissue has been reported [25]. In vivo, if heparin stimulates the production of chondroitin sulfate proteoglycans by vascular smooth muscle cells, it can play a potential role in the pathophysiology of atherosclerosis. This is because chondroitin sulfate proteoglycans, by virtue of their ability to form complexes with plasma apo B-containing lipoprotein [26], could facilitate lipid accumulation in the arterial wall. Acknowledgements This study was supported by grants HL-02942, HL42993 and HL 38844 from Tbe National Institutes of Health. References Berenson, G.S., Radhakrishnamurthy, B., Srinivasan, S.R., Vijayagopal, P., Dalferes, E.R., Jr. and Sharma, C., Recent advances in molecular pathology. Carbohydrateprotein macromolecules and arterial wall integrity - A role in atherogenesis, Exp. Mol. Pathol., 41 (1984) 267. Camejo, G., The interaction of lipids and lipoproteins with the intracellular matrix of arterial tissue, Adv. Lipid Res., 19 (1982) 1. Wight, T.N., Cell biology of arterial proteoglycans, Arteriosclerosis, 9 (1989) 1. Srinivasan, S.R., Dolan, P., Radhakrishnamurthy, B., Pargaonkar, P.S. and Berenson, G.S., Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions, B&him. Biophys. Acta, 388 (1975) 58. Berenson, G.S., Radhakrishnamurthy, B., Srinivasan, S.R., Vijayagopal, P. and Dalferes, E.R., Jr., Promechanisms teoglycans and potential related to atherosclerosis, Ann. N.Y. Acad. Sci., 454 (1984) 69. Wight, T.N. and Hascall, V.C., Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture, J. Cell Biol., 96 (1983) 167. Schmidt, A., Von Teutul, A. and Buddecke, E., Characterization of proteoglycans synthesized by cultured arterial smooth muscle cells of the rat, HoppeSeyler’s Z. Physiol. Chem., 365 (1984) 445. Wight, T.N., Potter-Perigo, S. and Auhnskas, T., Proteoglycans and vascular cell proliferation, Am. Rev. Resp. Dis., 140 (1989) 1132. Guyton, J.R., Rosenberg, R.D., Clowes, A.W. and Karnovsky, M.J., Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant heparin, Cir. Res., 46 (1980) 625.

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10 Hoover, R.L., Rosenberg, R.D., Haering, W. and Karnovsky, M.J., Inhibition of rat arterial smooth muscle cell proliferation by heparin. Part II In vitro studies, Cir. Res., 47 (1980) 578. 11 Lyon-Giordano, B., Conaway, H. and Kefalides, N.A., The effect of heparin on tibronectin and thrombospondin synthesis by human smooth muscle cells, Biochem. Biophys. Res. Commun., 148 (1987) 1264. 12 Tan, E.M.L., Levine, E., Sorger, T., Unger, G.A., Hacobian, N., Planck, B. and Ioxzo, R.V., Heparin and endothelial cell growth factor modulate collagen and proteoglycan production in human smooth muscle cells, Biochem. Biophys. Res. Commun., 163 (1989) 84. 13 Tan, E.M.L. Dodge, G.R., Sorger, T., Kovalszky, I., Unger, G.A., Yang, L., Levin, E.M. and Iozzo, R.V., Modulation of extracellular matrix gene expression by heparin and endothelial cell growth factor in human smooth muscle cells, Lab. Invest., 64 (1991) 474. 14 Ross, R., The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers, J. Cell Biol., 50 (1971) 172. 15 Edwards, I.J., Wagner, W.D. and Owens, R.T., Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells, Am. J. Pathol., 136 (1990) 609. 16 Hartree, E.F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. B&hem., 48 (1972) 422. 17 Carbon, D.M., Structure and immunochemicalproperties of oligosaccharides isolated from pig submaxillary mucins. J. Biol. Chem., 243 (1968) 616. 18 Gowda, D.C., Bhavanandan, V.P. and Davidson, E.A.,

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells, J. Biol. Chem., 261 (1986) 4926. 19 Saito, H., Yamagata, T. and Suzuki, S., Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates, J. Biol. Chem., 243 (1968) 1536. 20 Wasteson, A., A method for the determination of the molecular weight and molecular weight distribution of chondroitin sulfate, J. Chromatogr., 59 (1971) 87. 21 Srinivasan, S.R., Lopez-S.A., Radhakrishnamurthy, B. and Berenson, G.S., Complexes of serum pre-@- and /Ilipoproteins and acid mucopolysaccharides, Atherosclerosis, 12 (1970) 321. 22 Reilly, C., F&e, L. and Rosenberg, R.D., Heparin inhibition of smooth muscle cell proliferation: A cellular site of action, J. Cell. Physiol., 129 (1986) 11. 23 Nader, H.B., Buonassisi, V., Colbum, P. and Dietrich, C.P., Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells, J. Cell. Physiol., 140 (1989) 305. 24 Wight, T.N., Snow, A.D., Bolender, R.P., Lara, S., Fingerle, J., Certeza, S. and Clowes, A.W., Heparin influences the deposition of elastin, collagen and proteoglycans in the intimal extracellular matrix after arterial injury, J. Cell Biol., 107 (1989) 592a. 25 Ixuka, K. and Murata, K., Occurrence of heparin or its related acid glycosaminoglycan in human aortic tissue, 26

Experientia, 29 (1973) 655. Vijayagopal, P., Srinivasan, S.R., Radhakrishnamurthy, B. and Berenson, G.S., Interaction of serum lipoproteins and a proteoglycan from bovine aorta, J. Biol. Chem., 256 (1981) 8234.