Effect of calcium on cell proliferation and extracellular matrix synthesis in arterial smooth muscle cells and dermal fibroblasts

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

4,

307-317 (1986)

Effect of Calcium on Cell Proliferation and Extracellular Synthesis in Arterial Smooth Muscle Cells and Dermal Fi broblastsl BOHUMILAROKOSOVA~AND Department

of Biochemistry,

J.PETERBENTLEY

Oregon Health Sciences University, Road, Portland, Oregon 97201 Received

October

Matrix

3181 SW Sam Jackson

Park

18, 1985

The effect of calcium on cell proliferation and connective tissue formation was studied in cultured vascular smooth muscle cells (SMC) and dermal tibroblasts. Calcium deficiency caused a modest decrease in proliferation of smooth muscle cells but this effect was small compared to that previously observed with libroblasts. Synthesis of connective tissue components was affected differently in the two cell types. Biosynthesis of proteoglycans was assessed by metabolic labeling of their glycosaminoglycan side chains. Different levels of extracellular calcium did not affect proteoglycan production by fibroblasts, but it was significantly reduced in smooth muscle cells incubated in calcium-deficient medium. Both smooth muscle cells and fibroblasts were able to produce appreciable amounts of collagen in the complete absence of calcium and in both cell types collagen synthesis was increased when calcium was present. Fibroblasts, however, showed a much smaller response to calcium than did smooth muscle cells. In fibroblasts the maximum rate of collagen synthesis was achieved in a narrow range of calcium concentration which was slightly below that found commonly in the tissue culture medium. By contrast, in smooth muscle cells the rate of collagen synthesis increased greatly when calcium was present and this elevated rate persisted even when the cells were exposed to high levels of extracellular calcium. We conclude that these findings may be of significance to the development of atherosclerotic lesions. 0 1986 Academic Press, Inc.

INTRODUCTION Calcium has a well-defined role in many important physiological functions such as blood coagulation, muscle contraction, nerve impulse transmission, and mineralization of the skeletal matrix. In addition to directly forming the inorganic phase of the skeletal tissue, calcium has recently been shown to affect the synthesis of the organic matrix components by regulating the phenotypic expression of collagen types in the forming skeleton (Than and Lynch, 1983). Since calcium is also known to accumulate in the advanced atherosclerotic lesions, it seems important to determine whether changes in the environmental calcium concentration have any effect on synthetic activities of vascular smooth muscle cells. Smooth muscle cell hyperplasia and excessive extracellular matrix production are well-known features of atherosclerosis (see Ross and Glomset, 1976). Collagen is the most prominent of the extracellular components which accumulate in the lesion (Smith, 1974), but the stimulus for its increased synthesis is unknown. In a recent study (Rokosova ef al., 1986) we have found that in tissue specimens obtained from atherosclerotic human aortas, the rate of collagen synthesis was i Supported by National Institutes of Health Grant HL-14126 and by a grant from the Office of Naval Research (NOO14-84-KO402). 2 To whom correspondence should be addressed. 307 0014-4800/86 $3.00 Copyright Q 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

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higher in those tissue segments which had a higher calcium content. To determine whether this effect was indeed related to calcium, we examined cultured vascular smooth muscle cells and analyzed their responses to calcium. Since previous studies with fibroblasts (Balk et al., 1973; Boynton et al., 1977; McKeehan and Ham, 1978; Hazelton et al., 1979) have established that extracellular calcium has a regulatory effect on their proliferation in vitro, both cell proliferation and extracellular matrix formation were examined in our study. Data obtained with vascular smooth muscle cells were compared to those gathered with cultured tibroblasts. METHODS Cell Cultures and Cell Growth Assay Cultures of arterial smooth muscle cells were initiated from medial explants of bovine thoracic aortas by the procedure established by Ross (1971). Human dermal fibroblasts were grown from explants of neonatal foreskin from which the epidermal cells had been removed by gentle trypsinization (Hawley-Nelson et al., 1980). Both smooth muscle cells and tibroblasts were cultured either in Medium 199 or in McCoy’s 5a medium containing 10% fetal calf serum (HyClone Laboratories, Logan, Utah) and gentamycin. The concentration of calcium in the medium or its flux into the cells was manipulated in one of the following ways: (1) by addition of EGTA, which is a specific calcium chelator, to the growth medium (calcium contents of the medium and serum were both taken into the account); (2) by addition of calcium chloride to calcium-free medium (M-199, prepared by M.A. Bioproducts); (3) by adding to the culture medium calcium ionophore A23187 (Sigma) which increases transmembrane flux of calcium along a concentration gradient (Reed and Lardy, 1972). The cell proliferation was examined by measuring the DNA synthesis either after serum stimulation of growth-arrested preconfluent cells, or after inoculation of the cells into calcium-deficient medium to which various supplements of calcium chloride had been added. The specific conditions are described in legends to the tables and figures. [‘H]Thymidine incorporation into DNA was measured as described in detail previously (Rokosova and Bentley, 1980); cell numbers were determined with the fluorometric DNA assay (Lebarca and Paigen, 1980). The calcium content in the culture medium was determined by a complexometric titration (Connerty and Briggs, 1966). Each experimental condition was analyzed in triplicate cell cultures and the data are presented as means +- SD. All tissue culture products, unless stated otherwise, were purchased from Gibco Laboratories, other reagents were from Sigma Chemical Company. Assay of Proteoglycan Synthesis Proteoglycan biosynthesis was followed by labeling their glycosaminoglycan side chains. Confluent cultures were incubated in serum-free medium (specified in legends to figures and tables) with [3H]glucose (10 &i/ml medium) for 48 hr. Following incubation the culture medium was withdrawn, the cell layer washed with saline, and the wash added to the medium. The washed cells were trypsinized and separated from the trypsin-released material by centrifugation. The cell pellet served for the assay of DNA. The trypsin-solubilized material containing glycosaminoglycans associated with the cell surface was analyzed together with the cell medium. Carrier chondroitin sulfate (CSA) was added to the combined

EFFECT OF CALCIUM

ON CELL PROLIFERATION

309

material to a final concentration of 100 p&ml and the secreted proteoglycan-contaming material was precipitated by addition of 4 vol of ethanol at 4°C. The ethanol-precipitated material from the cell surface and the cell medium was recovered by centrifugation, suspended in a small amount of water, and subjected to digestion with papain to liberate the glycosaminoglycan side chains from proteoglycans. The conditions of papain digestion were described earlier (Bentley and Rokosova, 1970). After the digest was dialyzed and clarified by centrifugation, the liberated glycosaminoglycans were recovered by precipitation with ethanol, followed by reprecipitation with 1% cetylpyridinium chloride (CPC). This final precipitate was washed with water and redissolved in 1.25 M MgCl, which dissociates CPC-glycosaminoglycan complexes. Aliquots of the solubilized material were counted for radioactivity. Assay of Collagen Synthesis Collagen synthesis was assayed after the cells reached confluence. The growth medium was replaced with serum-free medium (specified in the appropriate legends of figures or tables) containing 100 p&ml ascorbic acid and 100 &ml @aminopropionitrile to prevent collagen crosslink formation. The cells were preincubated for 30 min in this medium before the addition of [3H]proline for metabolic labeling of collagen. The incubation was carried out for 24 hr and the labeled collagen formed was assayed by the method of Webster and Harvey (1979) with some modifications. In brief, the cells were grown on 25cm* tissue culture plates. After incubation, the cell cultures were acidified with acetic acid and digested with pepsin (50 pg/ml) for 16 hr at 4°C with gentle shaking. The medium was then withdrawn and the cells (which remained attached to the tissue culture plate) were washed with 0.5 M acetic acid. The cell wash was combined with the pepsin digest. The cell layer was covered with 1 ml of water and neutralized with 0.01 M NaOH, and the cells were released from the plate by trypsinization. The cellular collagen was then reextracted by an overnight incubation with pepsin under conditions similar to those described above. Following this, the cell debris was separated by centrifugation and the pellet analyzed for DNA content. The cell extract was combined with the pepsin digest of the cell medium. Carrier lathyritic rat skin collagen was added to the acetic acid-pepsin extract of cells and medium to give a final concentration of 100 pg collagen/ml. The collagens were then precipitated with 1.8 M sodium chloride. This step deviates from the original Webster and Harvey method in that a higher final salt concentration is used in order to ensure precipitation of the type IV and V collagens, if present. These collagens do not precipitate from acidic solutions at lower salt concentrations (Rhodes and Miller, 1978). The precipitated labeled collagen and the carrier were recovered at IOOOOg, dissolved in a small volume of 0.15 M NaCl at neutral pH, and reprecipitated at 4 M NaCl. The collagen pellet was then washed with 20% ethanol and dissolved in 0.5 M acetic acid, and aliquots were used for radioactivity counting in Aquasol liquid scintillation cocktail. RESULTS The Effect of Calcium on Smooth Muscle Cell Proliferation This experiment was performed in McCoy’s 5a medium in which the normal calcium chloride concentration is only 0.9 mM (as opposed to the other commonly used tissue culture media which contain 1.26 or 1.8 nuJ4 calcium chloride).

ROKOSOVA

310

AND BENTLEY

To reduce further the available calcium in the growth medium, we added 1.7 mM EGTA. This concentration was in excess of that needed to chelate all the calcium originally present in McCoy’s medium because addition of serum to the medium contributes additional calcium (Balk, 1971). Thus the “available free calcium” (Table I) is a sum of calcium content in the medium and serum plus that of added CaCI, from which calcium chelated by 1.7 mM EGTA had been subtracted. Microscopical examination of the cells grown in the medium containing EGTA did not reveal any changes in cell morphology. As could be expected, when the growth-arrested preconfluent smooth muscle cells were transferred into the growth medium (McCoy’s medium with 5% serum), a high rate of [3H]DNA synthesis was achieved (Table I). When calcium in the growth medium was completely chelated by addition of EGTA, the rate of growth recovery was reduced, but not completely inhibited. Addition of calcium chloride to the EGTA-treated medium progressively restored the synthetic rate of DNA to control levels. Thus smooth muscle cells differ from fibroblast, the growth of which is completely supressed at low levels of extracellular calcium or in EGTA-treated medium (Balk, 1971; Boynton and Whitfield, 1976). When smooth muscle cells were seeded in calcium-free Medium 199 with or without added calcium, the cells were able to proliferate even in the medium to which no calcium was added (Table II). However, since no EGTA was added, the growth medium contained calcium contributed by the 10% fetal calf serum (0.3 mM total calcium as determined by complexometric titration). Microscopic examination of cells seeded into this calcium-deficient medium revealed that they were slower in initiating proliferation; nevertheless the cell density attained after 3 days in culture was only slightly lower than that reached in the calcium-supplemented medium (Table II). It thus appears that in contrast to fibroblasts, in which proliferation is tightly controlled by the calcium levels in their medium (Balk, 1971; Dulbecco and Elkington, 1975; Boynton et al., 1977; McKeehan and Ham, 1978; Hazelton et al., 1979), calcium above 0.3 mM did not exhibit any significant regulatory effect upon the rate of smooth muscle cell proliferation. The concentration of calcium in the medium does not, of course, reflect intracellular concentrations. Therefore, to modulate the cytoplasmic concentration of TABLE I Effect of Calcium on DNA Synthesis by Arterial Smooth Muscle Cells Released from Growth Arrest Culture conditions Serum (%I 1 5 5 5 5 5

EGTA

+ + + +

CaCI, (mM)

Available free calcium hM)

1 2 3

0.93 1.05 None 0.35 1.35 2.35

[3H]Thymidine uptake (cpdpg DNA) 430 31,200 21,300 20,900 27,800 30,300

2 r 2 f k f

70 660 795 1020 1170 760

Note. Preconfluent smooth muscle cells, grown in McCoy’s Sa medium were arrested in growth by reducing the serum concentration to 1%. After 3 days this medium was replaced with a fresh growth medium containing 5% serum. EGTA (1.7 mA4) and various concentrations of calcium chloride (l-3 mM) were added to this growth medium. The cells were preincubated for 1 hr in the tested medium before 13H]thymidine labeling of the cells for 16 hr.

EFFECT OF CALCIUM

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ON CELL PROLIFERATION

TABLE II Effect of Calcium on Arterial Smooth Muscle Cell Growth Calcium addition to the growth medium (mM) None 0.1 0.5 1.0 1.5 1.8 2.0 2.5 3.0 5.0

DNA hhlaW 6.44 7.21 9.20 8.50 8.93 6.38 6.33 6.81 7.70 7.15

2 f 2 k f 2 + k 2 +

0.61 1.62 1.36 O.% 0.75 1.36 1.53 0.78 0.54 0.83

Note. Smooth muscle cells were inoculated into calcium-free medium (M-199) containing 10% fetal calf serum to which supplements of O-5 mM CaCl, were added. The initial cell density was 2.5 x 104 cells/cm* and density reached after 3 days in culture was determined by the DNA assay.

free calcium in the cultured cells, the ionophoretic antibiotic A23187 (Reed and Lardy, 1972) was used. Addition of this agent to the normal cell medium (containing calcium) did not induce any morphological changes in the cells at either concentration, but caused a marked inhibition of the [3H]DNA synthesis. This indicates that the profound rise in internal free calcium, as generated by the ionophore, was detrimental to the smooth muscle cell proliferation (Table III). That the ionophore itself was not toxic to the cells is attested by its effect of collagen synthesis (see below). Effect of Calcium

on Synthesis of Components

of the Extracellular

Matrix

Rroteoglycan and collagen synthesis was studied in arterial smooth muscle cells and in dermal fibroblasts. The different levels of calcium in the cell medium did not affect proteoglycan synthesis by fibroblasts to any significant account, but it was reduced almost to half in smooth muscle cells incubated in the calcium-deficient medium (Table IV). Similar reduction of proteoglycan synthesis was observed after chelating the calcium in the medium of smooth muscle cells (Table V). This assay was performed in Dulbecco’s serum-free medium in which the normal concentration of calcium chloride is 1.8 mM. When this calcium was complexed with 1.8 mM EGTA the proteoglycan synthesis was reduced to about [3H]Thymidine

TABLE III Uptake by Arterial Smooth Muscle Cells in the Presence of Calcium Ionophore Addition to growth medium None A23187 10 pM 20 FM

cpm/kg DNA 29,200 f 1052 10,200 2 880 4,900 + 570

Note. Growth-arrested smooth muscle cells (see legend to Table I) were returned to the McCoy’s growth medium containing 5% serum, to which calcium ionophore A23187 was added (lo-20 CLiM), and incubated with [sH]thymidine for 16 hr.

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Effect of Calcium on Glycosaminoglycan Calcium concentration in culture medium (W 0.0 0.25 0.5 1.0 1.5 2.0 3.0

AND BENTLEY

TABLE IV Synthesis by Dermal Fibroblasts and Arterial Smooth Muscle Cells [3H]Glycosaminoglycan Fibroblasts 960 1090 1095 1025 1147 987 1185

t f -t ? k k t

106 110 54 98 85 139 64

(cpm/ug DNA) Smooth muscle cells 1500 f 2800 f 2860 f 2200 -c 2300 f 2100 f

116 127 184 112 215 219

Note. Confluent cells were washed and the growth medium was replaced with serum-free calciumdeficient medium (M-199) to which various amounts of calcium chloride were added. After 1 hr preincubation, 10 lrJ.X/ml of [3H]glucose was added and the glycosaminoglycans were labeled by incubation for 48 hr.

half and the maximum rate of the glycosaminoglycan chain synthesis was not restored completely when CaCI, was added to the EGTA-treated cultures. We were unable to determine the calcium concentration which would restore the synthetic rate to the levels detected in the nonchelated 1.8 mit4 calcium in the normal medium, since additions of calcium chloride to the EGTA-treated serum-free medium at concentrations above 3 mM caused changes in the cell morphology. Nevertheless, the data (Table V) suggest that even though calcium may not be an absolute requirement for proteoglycan synthesis by the smooth muscle cells, proteoglycan synthesis can be affected by the extracellular calcium. Thus smooth muscle cells differ in this respect from Iibroblasts. Similar to the effect upon smooth muscle cell proliferation, addition of the calcium ionophore to the normal culture medium led to the inhibition of proteoglycan synthesis (Table V). The changes in the extracellular calcium concentration had a profound effect on collagen synthesis by the vascular smooth muscle cells. Data in Table V indicate that removal of calcium from the medium by chelation with EGTA caused a reduction of collagen synthesis to less than 40%. In contrast to the effect upon proteoglycan synthesis, addition of 1 mM CaCl, to the EGTA-treated medium TABLE V Effect of Calcium on Collagen and Glycosaminoglycan Synthesis by Arterial Smooth Muscle Cells Addition to medium None EGTA EGTA + 1 n&f CaCl, + 2 nut4 CaCl, + 3 mM CaCl, A23187 (10 p&f)

[3H]Glycosaminoglycan (cwbg DNA) 4600 f 90 2450 t 25 2750 f 30 3OOO-c50 2945 2 35 2350 k 45

[3H]Collagen (cm4.a DNA) 12,400 4,600 15,200 17,600 18,500 23,108

2 -c 2 f -c f

420 370 530 240 880 3150

Note. Confluent cell layers were washed and the growth medium was replaced with serum-free Dulbecco’s medium in which free calcium was blocked by addition of 1.8 m&f EGTA. The ionophore was added into unmodified medium. Glycosaminoglycans were labeled by incubation with 10 &i/ml of [3H]glucose for 48 hr; collagen was labeled by 24 hr incubation with 10 &i/ml of [3H]proline.

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restored the synthetic rate of collagen which was even greater than in the untreated control medium. To further study the effect of extracellular calcium upon the rate of collagen synthesis, we performed experiments with both cell types in calcium-free Medium 199 into which different concentrations of CaCl, were added. Data presented in Fig. 1 indicate that collagen synthesis in arterial smooth muscle cells was markedly stimulated by increased extracellular concentrations of calcium. Both smooth muscle cells and fibroblasts were able to produce significant amounts of collagen in the complete absence of calcium, but in both cell types collagen synthesis was increased when extracellular calcium was present. Fibroblasts, however, showed a different response to calcium than did smooth muscle cells. Fibroblasts appeared to have a narrower range of optimum calcium concentrations for maximal collagen synthesis (Fig. 1). By contrast, smooth muscle cells exhibited a much higher overall response to increased extracellular calcium. These cells were progressively stimulated by the increasing concentration of calcium, and the maximal rate of collagen synthesis in smooth muscle cells was achieved at higher calcium concentrations than in fibroblasts. In contrast to fibroblasts, this high rate of collagen synthesis by the smooth muscle cells persisted when the cells were exposed to the high extracellular calcium (up to 3 rnb4). In addition, collagen synthesis by the smooth muscle cells was the only synthetic parameter which we found to be stimulated when the level of intracellular free calcium was raised by the calcium ionophore (Table V). This stimulatory effect was, however, detected only when the inophore was added to the normal culture medium (unmodified Dulbecco’s medium) and it was abolished when A23187 was added to the medium enriched with additional calcium. This indicates that the cellular calcium overload achieved impaired the conditions required for maximal collagen synthesis.

15

0

01

Calcium

0.6

l.Ol.25

l.6 2.0 2.1 3.0 6.0

Concentration

(mM)

FIG. 1. Effect of extracellular calcium on collagen synthesis by vascular smooth muscle cells and by dermal fibroblasts. Confluent cell layers were washed and the growth medium was replaced with serum-free, calcium-free Medium 199 to which various amounts of calcium chloride were added. After 30 min preincubation, 10 @/ml of [sH]proline was added and the incubation carried out for 24 hr. Shaded blocks indicate collagen synthesis in fibroblasts; open blocks represent smooth muscle cells.

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DISCUSSION Although many previous studies of calcium effects on cell proliferation have been performed with fibroblasts (see below), studies addressing the role of calcium in various aspects of connective tissue formation are scarce (Gonnerman et al., 1976; Tajima et al., 1981, 1983; Oikarinen et al., 1984). This study examined the synthetic response of vascular smooth muscle cells to calcium and the results indicate that collagen and proteoglycan synthesis by these cells can be markedly affected by its extracellular concentration. In addition, we have found that smooth muscle cells differ from tibroblasts in their response to calcium. These effects were observed regardless of the type of tissue culture medium used or the mode by which calcium concentrations in the medium were manipulated. It is well established that untransformed fibroblasts in culture require calcium for their proliferation (Balk et al., 1973; Gail et al., 1973; Dulbecco and Elkington, 1975; Boynton and Whittield, 1976; McKeehan and Ham, 1978; Hazelton et al., 1979). Lowering extracellular calcium concentration blocks normal cells in the early G, phase of the cell cycle (Hazelton and lbpper, 1981) and thus calcium has been considered as one of the regulators of cell proliferation. The studies mentioned above were conducted with various types of cultured fibroblasts such as 3T3, WI-38, and chick embryo fibroblasts. Our data indicate that arterial smooth muscle cells differ from fibroblasts by their relative insensitivity to calcium with regard to their growth. Even though their proliferation was slightly stimulated by calcium, neither induction of growth in quiescent smooth muscle cells nor continuous propagation of these cells was calcium dependent (Table I and II). The cells grown in calcium-deficient medium did not differ morphologically from the cells grown in the “normal” medium, only their initial rate of proliferation was slightly slower. When calcium levels in the growth medium were raised above “normal,” no further growth stimulation was observed. Thus the idea that calcium might play a role in atherogenesis by stimulating smooth muscle cell proliferation is not supported by the experimental evidence. The overall rate of synthesis of proteoglycan did not appear to be affected by calcium in fibroblasts, but it was markedly reduced when smooth muscle cells were maintained in the calcium-deficient medium. Although we did not fractionate the extracellular proteoglycan secreted from the cells at various levels of extracellular calcium there is some evidence that the type of product can be affected. The study reported by Tajima et al. (1983) suggests that fibroblasts under “hypocalcemic” culture conditions produce less hyaluronic acid and more of the iduronic acid containing glycosaminoglycan (heparan sulfate and dermatan sulfate), whereas at increased calcium levels the ratio is reversed. Collagen synthesis was even more diversely affected when fibroblasts and smooth muscle cells were compared. Changes in calcium concentration in the cell medium profoundly affected the rate of collagen synthesis by the vascular cells, whereas the response of fibroblasts was of much smaller magnitude. In libroblasts we detected a higher rate of collagen synthesis at calcium concentrations below that found in commonly used tissue culture medium (MEM, Dulbecco, M-199) which contains above 1.25 mM calcium. We found the optimum to be 1.0-1.25 nut4 CaCI, and it is of interest that this concentration is very similar to the optimum calcium concentration reported by Tajima et al. (1981), even though the measurement of collagen production was done by different assays. In contrast to fibroblasts, collagen synthesis by the vascular smooth muscle cells was highly

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stimulated by increasing concentrations of the extracellular calcium and the maximal rate was retained at higher levels of extracellular calcium (1 J-3 n&f CaCl,). This implies that in smooth muscle cells high external calcium maintains the maximal rate of collagen synthesis. At present little is known about the overall control of collagen synthesis; thus it is impossible to speculate which synthetic signal would be augmented by the extracellular calcium. However, as the effect of the calcium ionophore A23187 (added to the normal medium) showed a profound stimulation of collagen synthesis by the smooth muscle cells, it not only appears that collagen synthesis was controlled by the extracellular calcium concentration, but also that changes in intracellular calcium had an effect. This agrees with findings of Dietrich and Duffield (1979) who showed that the calcium antagonist Verapamil (which reduces intracellular uptake of calcium) inhibited collagen synthesis in fetal rat bone. The intracellular calcium concentration depends ultimately on the entry of calcium into the cell. It is recognized that in mammalian cells a steady state of cytosol calcium level is maintained; however, changes in calcium influx are produced by ionic and hormonal stimuli and passive calcium entry is also a function of extracellular calcium concentration (Rasmussen, 1971; Moore and Pastan, 1978). At present, the cause of calcium accumulation in the developing atherosclerotic lesions is not clearly understood. It is known, however, that the final form of calcium deposits in the complicated lesions is calcium apatite (Schmid et al., 1980). Data gathered from this and our previous study (Rokosova et al., 1986) suggest that the involvement of calcium in the progression of atherosclerosis may be twofold. It seems reasonable to expect that besides being directly involved in the formation of the insoluble inorganic deposits in the vessel wall, calcium is also present in a soluble pool. Our data indicate that in this form calcium may stimulate the synthesis of the extracellular matrix laid down by the arterial smooth muscle cells. The physiological significance of this finding is underscored by several studies (Wartman et al., 1967; Whittington-Coleman et al., 1973; Hollander et al., 1979; Kramsch et al., 1980; Henry and Bentley, 1981; Ronleau et al., 1982) which demonstrate that agents preventing deposition of calcium in arteries or capable of blocking calcium flux into the vascular smooth muscle cells decreased the extent of experimentally induced atherosclerotic lesions without affecting serum hypercholesterolemia. Furthermore, hypertension-a well-identified risk factor for atherosclerosis -is reflected by elevated intracellular calcium levels (Zidek et al., 1982, 1983) and is also known to be associated with an increased rate of blood vessel collagen synthesis (Ooshima et al., 1974). REFERENCES BALK, S. (1971). Calcium as a regulator of the proliferation of normal but not of transformed chicken fibroblasts in a plasma-containing medium. Proc. Natl. Acad. Sci. USA 68, 271-275. BALK, S. D., WHITPIELD, J. F., YOUDALE, T., and BRAUN, A. C. (1973). Roles of calcium, serum, plasma and folic acid in the control of proliferation of normal and Rous sarcoma virus infected chicken tibroblasts. Proc. Natl. Acad. Sci. USA ‘IO, 675-679. BENTLEY, J. P., and ROKOSOVA, B. (1970). The metabolic heterogeneity of rabbit ear cartilage chondroitin sulphate. Eiochem. J. 116. 329-336. BOYNTON, A. L., and WHITFIELD, J. F. (1976). Different calcium requirements for proliferation of conditionally and unconditionally tumorigenic mouse cells. Proc. Natl. Acad.’ Sci. USA 73, 1651-1654. BOYNTON, A. L., WHITHELD, J. F., ISAACS, R., and TREMBLAY, R. (1977). The control of human

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RHODES, R. K., and MILLER, E. J. (1978). Physicochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17, 3442-3448. ROKOSOVA, B., and BENTLEY, J. P (1980). The effect of lysosomal enzymes on the proliferation of aortic smooth muscle cells and tibroblasts. Pathol. Biol. 28, 493-500. ROKOSOVA, B., RAPP, J. H., PORTER, J. M., and BENTLEY, J. P (1986). The composition and metabolism of symptomatic distal aortic plaque. J. Vast. Surg. 3, 617-622. RONLEAU, J. L., PARMLEY, W. W., STEVENS, J., WIKMANN-COFFELT, J., SIEVERS, R., MABLEY, R., and HAVEL, R. J. (1982). Verapamil suppresses atherosclerosis in cholesterol fed rabbits. Amer. J. Cardiol. 49,(Suppl. IV, Pt. I), 889. Ross, R. (1971)., The smooth muscle cell. II. Growth of smooth muscle cells in culture and formation of elastic fibers. J. Cell Biol. 50, 172-186. Ross, R., and GLOMSET, J. A. (1976). The pathogenesis of atherosclerosis. N. Eng. J. Med. 295, 369-377.

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