Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Experimental

Cell Research 166 (1986) 416-426

Synthesis of Sulfated Proteoglycans throughout the Cell Cycle in Smooth Muscle Cells from Pig Aorta MONIQUE BRETON, ELIANE BERROU, M.-CHRISTINE BRAHIMI-HORN, ELISABETH DEUDON and JACQUES

PICARD*

Laboratoire de Biochimie, INSERM U.181, Facultt! de Mkdecine Saint-Antoine, 75571 Paris Cedex 12, France

Cultured smooth muscle cells from pig aorta arrested in GO phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in Gl phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30-32 h; 75 % of these cells multiplied after 30-32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [35S]sulfate. Smooth muscle cells in Gl or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [35S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during Gl phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CLdB columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied: 1. [35S]Proteoglycan complexes represented at the end of Gl phase and of the cell cycle respectively 19 and 16% of the total [3sS]proteoglycans secreted into the medium. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k., after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k,, 0.68 at the end of Gl phase and 0.65 at the end of full cell cycle). 3. About 95 % of monomers synthesized at the two stages of the cell cycle were eluted at k,, 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the mediumduring the Gl and G2 phases. @ 19136Academic press, I~C.

Smooth muscle cells appear to play an important role in the development of atherosclerosis. In normal arteries, the smooth muscle cells are separated from the blood flow by a monolayer of endothelial cells and have a very low mitotic rate. By contrast, the initiation of the atherosclerotic plaque arises from the

* To whom offprint requests should be sent. Address: Laboratoire de Biochimie, INSERM U.181, Fact& de Mtdecine Saint-Antoine, 27 rue Chaligny, 755 71 Paris, Cedex 12, France. Copyrieht 0 1986 by Academic Fnss, Inc. All Ii&S of npr0duCtiOn in any form rescrvcd 0014-4827/86 so3.00

Sulfated proteoglycan

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migration and proliferation of smooth muscle cells, following rupture of the endothelial layer [l] and leads to a modification in the permeability of the intima to plasma components. This modification in the growth of smooth muscle cells is accompanied by changes in their metabolism which bring about increased deposition of components of the extracellular matrix, especially collagen and proteoglycan [2, 31. Various studies have shown that arterial smooth muscle cells in culture retain their differentiated function with respect to proteoglycan synthesis [4, 51. Moreover, an increase in the synthesis of proteoglycan (40-60%) was observed when primate arterial smooth muscle cells in culture were stimulated to divide [6]. Various studies with cultured chondrocytes, major proteoglycan synthesizing cells, have also shown that the length of time during which these cells remain in the Gl phase can influence the rate of sulfated proteoglycan synthesis [7, 81. Few studies have investigated the cell-cycle dependence of proteoglycan synthesis. An early study by Kraemer & Tobey [9] demonstrated a premitotic loss of surface heparan sulfate from synchronized Chinese hamster ovary (CHO) cells. More recently, it was shown that the rate of sulfated glycosaminoglycan synthesis was correlated with the rounding up of mitotic cells and their reattachment after cell division [lo, 111. The results concerning cells involved in secreting important quantities of proteoglycans into the extracellular medium [ 12, 131 indicated that hyaluronic acid, chondroitin sulfate and heparan sulfate were synthesized at various times during the cell cycle. However, heparan sulfate was not secreted into the medium, or else it was secreted in a modified form during the G2/M phase [12]. The present study was undertaken (1) to estimate the duration of the different cell-cycle phases of smooth muscle cells; (2) to investigate the rate of synthesis of proteoglycans produced by synchronized smooth muscle cells in dividing and in quiescent states; (3) to examine structural characteristics of proteoglycans secreted into the medium throughout a single cell cycle. MATERIAL

AND METHODS

Cell Culture Primary cultures of smooth muscle cells were obtained from media explants of pig aorta by a method similar to that employed by Ross [14]. Cells were cultured as previously described 1151in Dulbecco’s modified Eagle minimum medium (DMEM) supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 ug/ml streptomycin and 10% fetal calf serum (FCS) (KC Biological, Inc.) (together referred to as growth medium). For subcultures, cells were trypsinized with 0.25 % (w/v) trypsin (KC Biological, Inc.) for 10 min at 37°C. For experiments, cells in the 2-6 passage were plated in 25 cm2 flasks (Coming Glass Works, Corning, N.Y.), at a density of 12000 cells/cm* in 5 ml of growth medium for 72 h with a change of medium after 24 h. In order to establish quiescence, the growth medium was removed, the cell layer washed twice with serum-free medium and incubated for 5 days, with a change after 2 days, to serumfree medium supplemented with 0.1% (w/v) albumin (essentially fatty acid-free ,bovine serum, Sigma Chemical Co.) (referred to as basal medium). Cells which had stopped cycling in Gl or G2 are said to reside respectively in GO-G1 or GO-G2 [16]. Experiments were then initiated by refeeding with basal or growth medium. Exp Ceil Res 166 (1986)

418 Breton et al.

Growth Experiments DNA synthesis was measured as radio-isotopic thymidine incorporation into acid-precipitable material. The cells were pulsed during the last 60 min of the culture period with 0.5 @i/ml [methyl3H]thymidine (25 Wmmol, Amersham International Ltd). At the end of the labelling period, the radioactive medium was removed. The cell layer was washed gently five times with 5 ml cold Dulbecco’s phosphate-buffered saline (PBS), twice with 5 ml cold 10% trichloroacetic acid (TCA) containing 10 mM thymidine for 15 min at 4°C and twice with 5 ml ethanol. The dried, fixed cells were dissolved in 0.5 M NaOH, an aliquot was added to Biofluor (New England Nuclear) after neutralization and the radioactivity was measured in an Intertechnique scintillation counter. Cell numbers were determined at the end of each labelling period with a cell counter (OrthoInstrument).

Measurement of the Rate of 35S-Labelled Proteoglycan Synthesis Experiments concerning proteoglycan synthesis were initiated by incubation of quiescent cells in basal or growth mediums containing 10 @i/ml Na2[35S0~-] (100 mCi/mmol, Amersham Intemational Ltd). At the end of the different periods of time up to 60 h, the medium was poured off and the cell layer washed and fixed as described for [3H]thymidine incorporation but with a TCA solution containing 0.1 M sodium sulfate. The cell layer was dissolved in 0.5 M NaOH and the radioactivity of aliquots measured in Biofluor. Guanidine-HCl powder was added to a 1 ml aliquot of each medium sample to obtain a concentration of 4 M. 0.5 ml of each of these aliquots was then applied to a separate Sephadex G-25 M, PD-10 column (Pharmacia Fine Chemicals). The columns were eluted with 4 M guanidine-HCl, 0.05 M sodium acetate solution containing 0.1 M sodium sulfate. Chromatography on PD-10 columns provided a good separation of 35S-labelled macromolecules (eluted in the column void volume) from the unincorporated precursor (eluted in the retarded fractions) with recoveries of the total radioactivity superior to 95 %.

Relative Hydrodynamic Size of 35S-Labelled Proteoglycans At the end of different label@ periods, the medium was removed and protease inhibitors were added to obtain 10 mM sodium EDTA, 0.1 M 6-amino hexanoic-acid, 5 mM benzamidine hydrochloride, 0.001% (w/v) soybean trypsin inhibitor and 0.2 mM phenylmethyl sulfonyl fluoride. After dialysis at 4°C against a 0.1 M ammonium sulfate solution and then 50 mM sodium acetate buffer both containing protease inhibitors, 3SS-labelled proteoglycans were precipitated with cetylpyridinium chloride and ethanol as previously described [4]. The hydrodynamic sizes of the radiolabelled proteoglycans were investigated by chromatography on Sepharose CL-2B and CLdB gels (Pharmacia Fine Chemicals) at 4°C. Sepharose CL-2B columns (1.6~100 cm, flow rate 8-9 mlih, 2.4-2.6 ml fractions) and Sepharose CLdB columns (0.9~100 cm, flow rate 6-7 ml/h, 0.8 ml fractions) were eluted under the associative conditions of 0.5 M sodium acetate. Sepharose CL-6B columns (0.9~60 cm, flow rate 5-6 ml/h, 0.5 ml fractions) were also eluted under the dissociative conditions of 4 M guanidine-HCl, 50 mM sodium acetate, pH 5.8. Column recoveries ranged from 75 to 85 % of the total applied radioactivity with a higher yield for chromatography under dissociative conditions than under associative conditions.

Characterization of Glycosaminoglycans of 3’S-Labelled Proteoglycans Secreted into the Medium Proteoglycans secreted into the culture medium were precipitated with ethanol prior to proteolytic digestion with pronase 1171.The glycosaminoglycans were then purified by alternate CPC-ethanol precipitations and separated by electrophoresis on acetate cellulose strips as previously described 1171. The proteoglycan carbohydrate chains were characterized by differential susceptibility to glycosaminoglycan degrading enzymes: bovine testicular hyaluronidase (EC 3.2.1.35, Sigma) and chondroitin ABC lyase (Proteus vulgaris, EC 4.2.2.4, Miles Scientific) as previously described [4]. Specific degradation of heparan sulfate with HN02 was carried out according to Lindahl et al. [ItI]. Exp CellRes 166(1986)

Sulfated proteoglycan synthesis throughout the cell cycle 419 RESULTS Cell Replication When smooth muscle cells are cultured in basal medium, a viable but nonproliferative cell population results. More than 95% of these cells excluded trypan blue, and the plating efficiency determined 8 h after trypsinization was 90-95 %. The effect of the addition of 10% FCS to quiescent smooth muscle cells on DNA synthesis and cell multiplication is shown in fig. 1. The incorporation of 13H]thymidine into DNA started 18 h after replacing basal medium by growth medium and attained a maximum at about 24 h, the value of which was equal to more than 130-fold that at 16 h. Incorporation then decreased rapidly and at 32 h was again equal to the value at 18 h. The addition of serum often (but not always) induced, in less than 3 h, an increase in the cell number (about 20%) without incorporation of [3H]thymidine into DNA (fig. 1). As demonstrated by the single [‘Hlthymidine peak, this increase represented cells which had been blocked in the GO-G2 phase and which joined in the Gl phase the majority of cells which had been blocked in GO-G1 , The number of cells detected at 3 h after initiation of the experiment by serum addition was taken as a reference in the estimation of the proportion of cells which divided during the cell cycle. About 35 % of the cells divided 68 h after maximal DNA synthesis, and 14 h after this maximal synthesis 66% of the cells had multiplied. Subsequently, this proportion did not increase significantly. A new change of medium with growth medium performed 42 h after the initiation of the experiment provoked a rapid increase in the number of cells. This increase corresponded to the division of 70% of the cells which had been arrested in their growth. Based on the data for [3H]thymidine incorporation and cell counts, it may be concluded that 90% of smooth muscle cells were in the Gl phase for the period O-16 h after addition of serum, they then advanced into the S phase after 18 h and progressively entered the G2 phase after 24 h. Mitosis took place between 30 and 38 h. When the experiment was initiated by exposing quiescent cells to basal medium, the incorporation of [3H]thymidine was about 1500 cpm/106 cells irrespective of the incubation time and the number of cells was not modified significantly at least until 60 h. Kinetics of Incorporation of [35S]Sulfate into Cell Layer and Medium Macromolecules As observed by Chang et al. [19] with confluent monkey smooth muscle cells, [35S]sulfate incorporation was shown to be specific for the labelling of proteoglycans synthesized by pig smooth muscle cells. The [35Slsulfate incorporated into macromolecular material at the end of Gl and G2 phases and at the end of the cell cycle was totally liberated after treatment with chondroitin ABC lyase and i%p Cell Res 166 (1986)

420 Breton et al. -1.25

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Fig. 1. Proliferation of pig aorta smooth muscle cells after release from CO. Cells were growtharrested by serum deprivation for 96 h. At r=O, the cells were released from the GO block by placing them in growth medium. At the indicated times after release, cell numbers (A- --A) were determined with a cell counter and [“Hlthymidine incorporation (A-A) into DNA during the last 60 min of the culture period was measured. DNA was precipitated with TCA and processed for scintillation counting as described in Materials and Methods. The number of cells were also determined after a new change of medium with growth medium performed 42 h after the initiation of the experiment
Erp Cell Res 166 (1986)

Sulfated proteoglycan

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nitrous acid treatments which degrade chondroitin sulfate- dermatan sulfate and heparan sulfate respectively (data not shown). When quiescent cells were exposed to medium supplemented with 10% FCS, the incorporation of [35S]sulfate into cell layer proteoglycans (fig. 2) increased linearly for up to 20 h, that is while cells were in the Gl phase. Between 20 and 24 h the values did not vary significantly, the pool of [35S]proteoglycans thus appeared to remain constant while the majority of the cells were in S phase. Between 24 and 30 h, the incorporation of the labelled precursor increased by about 25%. There was thus renewed synthesis of [35S]proteoglycans at the end of S phase and/or during G2 phase. At the moment when the cells began to divide, between 30 and 32 h after the initiation of the experiment, [35S]sulfate incorporation stopped, represented by the 35 % decrease in the incorporation of precursor relative to the total cell number. Moreover, while certain cells were dividing, others were already in the Gl phase and a renewed increase in the incorporation of [35S]sulfate was observed. The kinetics of incorporation of radioactive precursor into proteoglycans liberated into the medium (fig. 2) was similar to that for proteoglycans of the cell layer. The incorporation of [35S]sulfate increased while cells were in the Gl and G2 phases and stopped at the beginning of S phase and during mitosis. However, it was noted that during the first third of the Gl phase, the synthesis and/or liberation of [35S]proteoglycans appeared to be slower than during the remainder of this phase. Under the labelling conditions employed, the radioactive proteoglycans first appeared in the medium, 40 min after initiation of the experiments. When quiescent cells were re-placed into fresh basal medium, a substantial increase in synthesis of [35S]proteoglycans was observed (fig. 3). Comparison of results obtained when cells were stimulated to divide shows that the incorporation of [35S]sulfate into cell layer or medium proteoglycans was similar during the first 6 h for both activated and quiescent populations; for longer labelling periods, incorporation always remained lower in cells maintained in a quiescent state. Analysis of Extracellular

Proteoglycans

The hydrodynamic size distribution of proteoglycans liberated into the medium was determined at two different moments in the cell cycle: at the end of the Gl phase (fig. 4a) and at the end of the cell cycle (fig. 4 b), i.e., respectively 20 and 35 h after initiation of the experiments. Chromatography on Sepharose CL-2B under associative conditions of [35S]proteoglycans liberated into the medium at 20 or at 35 h resulted in the fractionation of two populations of proteoglycans: one excluded from the column (peak A) representing about 20% of the total eluted radioactivity and the other included in column fractions (peak B) at a k,, of 0.76. In order to determine the proportion of [35S]proteoglycans present in the medium in a complex form, the [35S]proteoglycans of peak A were rechromatographed on Sepharose CL-2B under dissociative conditions. The resulting elution profiles for [35S]proteoglycans synthesized at 20 and 35 h were similar. They Exp Cell Res 166 (1986)

422 Breton et al. a: 20 hours

KAV Sapharos.

CL-2B

b: 35 hours

Sapharos.

CL-28

Sepharona

CL-26

KAV Sapharosa

CL-66

Fig. 4. Hydrodynamic size distribution of 35S-labelled proteoglycans secreted into the medium during

two different periods of the cell cycle. At r=O, quiescent smooth muscle cells were supplied with growth medium containing [35S]sulfate. Radiolabelled proteoglycans secreted into (a) the medium during Gl phase (20 h); (&) an entire cell cycle (35 h) were first chromatographed on Sepharose CL-2B under the associative conditions of 0.5 M sodium acetate (-). Afterwards, [3sS]proteoglycans of excluded fractions (peak A) were rechromatographed on Sepharose CL-2B column under the dissociative conditions of 4 M guanidinaHC1,50 m&l sodium acetate (- - - -) and [35S]proteoglycans of retarded fractions (peak B) were re-run on Sepharose CL-6B column under associative conditions. [3’S]Proteoglycans secreted into the medium during an entire cell cycle and obtained in retarded fractions of the dissociable Sepharose CL-2B column (peak A-2) and [‘%]proteoglycans of peak B were rechromatographed on Sepharose CL-6B column under dissociative conditions.

Exp Cell Res 166 (1986)

Sulfated proteoglycan

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423

showed two peaks, one excluded (peak A-l) representing 6-7 % of the total eluted radioactivity and the other retained (peak A-2) at a k,, of 0.65. The medium [3’Slproteoglycans were present in a dissociable complex form and represented at 20 and 35 h respectively 19 and 16% of the total [35S]proteoglycans secreted into the medium. Proteoglycans in peak A-2 obtained after 35 h incorporation were almost totally excluded from a Sepharose CL-6B column eluted under dissociative conditions. [35S]Proteoglycans in peak B were rechromatographed on Sepharose CL-6B under associative conditions. Regardless of the phase of the cell cycle during which the proteoglycans were synthesized, about 5% of the radioactivity was eluted in the column void volume, the remainder was eluted at a k,, of 0.25. [35S]Proteoglycans in peak B obtained after 35 h incorporation were also rechromatographed under dissociative conditions on Sepharose CL-6B. 35 % of the radioactivity was eluted in excluded fractions and the remainder at a k,, of 0.13. Thus the [35S]proteoglycans of peak B were monomers and in fact appeared to have a slightly higher relative hydrodynamic size when eluted under dissociative rather than associative conditions. The glycosaminoglycan composition of the total proteoglycans secreted into the medium at 20 and at 35 h as well as that for the monomer population (peak B) is shown in table 1. The glycosaminoglycan distribution of proteoglycans present in the medium in a complex form (peak A-2) was not examined because of the small amount available. This distribution did not seem to vary with the periods of the cell cycle that were studied. The chondroitin sulfate and dermatan sulfate chains of secreted proteoglycans present in the medium represented about 70% of the total glycosaminoglycans. This proportion did not change significantly for proteoglycans retained on a Sepharose CL-2B column. The proportion of hyaluronic acid secreted into the medium was higher than that for the tissue [4]. Also, the monomer population of the medium contained a higher proportion of hyaluronic acid than monomer proteoglycans extracted from the tissue.

Table 1. Glycosaminoglycan

distribution

of extracellular

HA

HS

CS-DS

20 h

Total proteoglycans Monomers

23 19

10 5

67 76

35 h

Total proteoglycans Monomers

20 21

11

69

7

72

proteoglycans

At t=O, quiescent pig aorta smooth muscle cells were stimulated to proliferate by placing them in growth medium. The relative distribution of the different glycossminoglycans: hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate-dermatan sulfate (CS-DS) of proteoglycans secreted into the medium during Gl phase (20 h) or an entire cell cycle (35 h) was determined by electrophoresis, as described in Materials and Methods. Results are expressed as a percentage of the total glycosaminoglycans isolated either from total proteoglycans or from the monomer population obtained by chromatography on Sepharose CL-2B column under associative conditions (see fig. 4, peak B). 28-868340

Exp Cell Res 166 (1986)

424 Breton et al. DISCUSSION Cultured arterial smooth muscle cells maintained for 5 days in medium supplemented with albumin became arrested in a reversible manner in the GO phase. Studies into DNA replication and proliferation of smooth muscle cells enabled firstly the demonstration of the synchronization of 90% of the cells, principally during the Gl and S phases, and secondly the determination, with relative precision, of the duration of the different cell cycle phases. The different methods used to arrest smooth muscle cells in the GO phase, including incubation in the presence of serum-deficient medium containing 2 or 5% of the platelet-poor plasma [20, 211, did not appear to modify the kinetics of incorporation of [3H]thymidine into DNA: incorporation commenced 16-18 h after replacing the medium of the quiescent cells with medium enriched in serum, and reached a maximum after 24-28 h. Thus incubation of smooth muscle cells in medium deficient in serum does not seem to modify their progression through the cycle when they are again incubated in growth medium. This method thereby permits examination of the direct effect of growth factors, hormones or any other factors on different metabolic pathways. These studies provide detailed information concerning the synthesis of sulfated proteoglycans by smooth muscle cells during their cell cycle. The main results of the cell-cycle dependence of sulfated proteoglycans were obtained either without discrimination between early, middle or late stages of Gl or G2+M [9, 10, 12, 221 or for the precise stages: late G2, M, and early Gl but in isolation [I I]. Moreover, although Wight has recently observed an increase of the [35S]sulfate incorporation in proteoglycans of the cell layer or secreted into the medium at 24 h after growth stimulation of quiescent smooth muscle cells [3], no study, to our knowledge, has investigated the possible influence of the entire cell cycle on the sulfated proteoglycan synthesis by smooth muscle cells. Our results show that the synthesis of sulfated proteoglycans of the cell layer and secreted into the medium stopped at the commencement of the S phase (from 20 to 24 h after replacing the basal medium of the quiescent cells with the growth medium) and during mitosis. On the contrary, cells in the Gl or G2 phases synthesized and secreted sulfated proteoglycans with a possible slow-down at the end of the G2 phase: the incorporation of [35S]sulfate into proteoglycan stopped when 55 % of the cells were either at the end of the G2 phase and ready to divide or were blocked at the end of the G2 phase due to a depletion of nutrients. Nevertheless, the decrease in available nutrients could not be responsible for the stoppage in synthesis of sulfated proteoglycans, since incorporation of precursor into these macromolecules was again observed 4 h after the beginning of cell division. The synthesis of sulfated proteoglycans during the Gl phase followed two different kinetics for proteoglycans of the cell layer and for proteoglycans secreted into the medium. The rate of incorporation of [35S]sulfate into proteoglycans of the cell layer remained constant during the GO-G1 passage and throughout the Gl phase. On the contrary, 6-8 h after having stimulated cell growth, the rate of tip

cell Res 166 (1986)

Sulfated proteoglycan

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appearance of sulfated proteoglycans in the medium increased. This could be a result of an acceleration either in the rate of synthesis of proteoglycans destined to be secreted or in its rate of liberation into the medium. However, since the rate of synthesis of sulfated proteoglycans retained in the cell layer did not vary during the Gl phase, it is probable that the secreted proteoglycans did increase at some point within Gl of the cell cycle. This study does not answer the question as to whether this point corresponds to the GO-G1 passage or to a particular step in the Gl phase. Nevertheless, Larsson et al. recently showed that fibroblasts in the GO phase require a preparatory period of 8 h before continuing to Gl [23]. Also, placing quiescent smooth muscle cells in either fresh growth medium or fresh basal medium did not influence significantly the rate of incorporation of [35S]su1fate into proteoglycans during at least the first 6 h but brings about considerable differences in the kinetics of incorporation of precursor 6-8 h after changing the medium. A few studies have examined the glycosaminoglycan composition of proteoglycans synthesized during the cell cycle. However, the degree of aggregation and the hydrodynamic size of proteoglycans either capable of forming complexes or present in a monomeric form has not been examined. Our results demonstrate that proteoglycans secreted into the medium during the cell cycle did not exhibit differences in the above structural characteristics to those synthesized during the Gl phase. Thus during the G2 phase, another period of substantial proteoglycan synthesis, arterial smooth muscle cells do not appear to synthesize distinct populations of proteoglycans destined to be secreted into the medium. In addition, the extracellular proteoglycan synthesized by cultured smooth muscle cells exhibits similar physico-chemical properties to those isolated from the arterial tissue [24]. However, monomer proteoglycans secreted into the medium possess a higher hydrodynamic size than those obtained from the aorta. This could result from a greater degradation of proteoglycans which remain in the extracellular matrix for a relatively longer time in comparison with those secreted into the medium. In fact, studies performed by Buckwalter & Rosenberg [25] with proteoglycans isolated from mature cartilage, have shown that the chondroitin-sulfate-rich region of the protein core exhibits variable sizes which may result from a proteolytic attack of this region of the protein core. These variations in the length of the protein core were not observed for proteoglycans synthesized by cultured chondrocytes from normal [26] or chondrosarcoma 1271tissue. In conclusion, the findings presented here showed that smooth muscle cells in viable but non-proliferative state which is also the case of these cells in a healthy aorta, synthesized sulfated proteoglycans and that the stimulation of smooth muscle cell proliferation induced an increase in the rate of sulfated proteoglycan synthesis which essentially occurred when cells traversed Gl and G2 phases. These observations are consistent with the main processes leading to the early development of atherosclerotic lesions. Exp Cell Res 166 (1986)

426 Breton et al. This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (INSERM, U.181) and the Fondation pour la Recherche Mtdicale. The authors thank Betty Jacquin for secretarial assistance.

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Ross, R & Glomset, J A, Science 180 (1973) 1332. Pietilti, K C Nikkari, T, Med biol61 (1983) 31. Wight, TN, Fed proc 44 (1985) 381. Horn, M C, Breton, M, Deudon, E, Berrou, E & Picard, J, Biochim biophys acta 755 (1983) 95. Wight, T N 62 Hascall, V C, J cell biol % (1983) 167. Wight, T N, Hascall, V C & Ross, R, J cell bio187 (1980) 119. Malemud, C J & Sokoloff, L, J cell physio184 (1974) 171. Miller, R P, Husain, M L Lohin, S, J cell physiol 100 (1979) 63. Kraemer, P H & Tobey, R A, J cell biol 55 (1972) 713. Blair, 0 C & Sartorelli, A C, Cytometry 5 (1984) 281. Preston, S F, Regula, C S, Sager, P R, Pearson, C B, Daniels, L S, Brown, P A & Berlin, R D, J cell biol 101 (1985) 1086. 12. Davidson, E A & MacPherson, I, Exp cell res 95 (1975) 218. 13. Otto, A M & Milhadt, P F, J supramol struct 13 (1980) 281. 14. Ross, R, J cell biol 50 (1971) 172. 15. Deudon, E, Breton, M, Berrou, E & Picard, J, Biochimie 62 (1980) 811. 16. Srinivasan, B D & Harding, C V, Invest ophthalmol4 (1%5) 452. 17. Picard, J, Paul-Gardais, A & Vedel, M, Biochim biophys acta 320 (1973) 427. 18. Lindahl, U, Backstrom, G, Jansson, L & Hallen, A, J biol them 248 (1973) 7234. 19. Chang, Y, Yanagishita, M, Hascall, V C & Wight, T N, J biol them 258 (1983) 5679. 20. Franks, D J, Plamondon, J & Hame, P, J cell physiol 119 (1984) 41. 21. Castellot, J J, Jr, Cochran, D L & Kamovsky, M J, J ceil physiol 124 (1985) 21. 22. Blair, 0 C, Burger, D E & Sartorelli, A C, Cytometry 3 (1982) 166. 23. Larson, 0, Zetterberg, A & Engstrom, W, J cell sci 73 (1985) 375. 24. Breton, M, Picard, J & Berrou, E, Biochimie 63 (1981) 515. 25. Buckwalter, J A & Rosenberg, L C, J biol them 257 (1982) 9830. 26. Mitchell, D & Hardingham, T, Biochem j 1% (1981) 521. 27. Fellini, S A, Kimura, J H & Hascall, V C, J biol them 256 (1981) 7883. Received February 7, 1986

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