21 (1975) 3 15-324

Atherosclerosis,

0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

EFFECT

OF

COLLAGEN

T. RijNNEMAA, Department

HYPERLIPIDEMIC BY CHICK

K. JUVA

of Medical

EMBRYO

SERUM

ON

THE

SYNTHESIS

OF

FIBROBLASTS

E. KULONEN

AND

Chemistry,

RAT

315

University

of Turku, SF-20520

Turku 52 (Finland)

(Received, revised, July Sth, 1974) (Accepted November 29th, 1974)

SUMMARY

The effect of hyperlipidemic rat serum and its fractions on the synthetic functions of embryonic fibroblasts was studied. Moderately hypercholesterolemic sera (100-140 mg/lOO ml) stimulated the synthesis of collagen, but not the synthesis of non-collagenous proteins nor the incorporation of glucosamine or cytidine. The stimulating principle was nondialyzable. It was not associated with the isolated total lipoproteins but was found in the infranatant fraction of sera centrifuged at a density of 1.220.

Key words : Collagen synthesis - Connective tissue activation - Dietary hyperlipidemia - Fibroblasts - [sH]Cytidine - j3HjGlucosamine - j3H]ProIine Serum cholesterol

INTRODUCTION

Descriptions of classic atherosclerotic lesions emphasize the accumulation of lipid material, connective tissue components, calcium and cell debris. Various mechanisms have been proposed for the primary injury of the arterial walll, but little is known about the pathogenesis at the molecular level. The thickening of the arterial intima is caused by the proliferation of mesenchymal cells and the accumulation of extracellular matrix. Although there are numerous reports on the role of lipids in promoting this proces9, the direct effect of hyperlipidemic serum on connective tissue cells has not been widely studied. The positive correlation between plasma cholesterol concentration and collagen synthesis in experimental granulation tissue of rat has

This work was supported by an institutional Life Assurance Companies.

research contract with the Finnish Association of

316

T. RijNNEMAA,

recently

been elaborated

by Pelliniemi3

stimulatory effect of hyperlipidemic tissue cells isolated from embryonic MATERIAL

AND

from our laboratory.

In this paper we report a

serum and of one of its fractions tendons.

were obtained

Muna Oy, Turku, Finland). The isolation ing to the method of Dehm and ProckopJ. embryos

E. KULONEN

on connective

METHODS

Isolation of tendon cells White Leghorn chick embryos

chick

K. JUVA,

were placed

in siliconized

from

a local hatchery

(Turun

of the cells was carried out chiefly accordThe leg tendons of about 20 16-17-day old glass vessels containing

3 ml modified

Krebs-Ringer medium (141 mM NaCl, 5.6 mM KCl, 3.0 mM CaClz, 1.4 mM KHZPO4, 1.4 mM MgS04, 22 mM glucose) buffered with 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES, Sigma), pH 7.4, 0.33 ml of 5% trypsin

(Type III, Sigma) in 0.154 M NaCl and 3.3 mg bacterial

collagenase

(Type I,

Sigma). The tendons were incubated at 37°C for 60 min with vigorous shaking. After incubation the samples were filtered through lens paper and the filtrates were centrifuged at 50 x g for 12 min at room temperature in 25-ml polycarbonate tubes. The supernatant was discarded and the cells were suspended in 6 ml of 0.1% soybean trypsin inhibitor (Serva Feinbiochemica, Heidelberg, Germany) in the modified Krebs-Ringer medium. The cells were washed twice with the same solution by centrifugation. Incubation with labelled precursors The isolated cells were suspended in modified Krebs-Ringer medium containing 0.015 % bovine serum albumin (A 4503, Sigma). Aliquots containing about lo6 cells in 3 ml of the medium were placed in 25-ml polycarbonate tubes which contained serum or one of its fractions (ride infia). After a 15-min preincubation at 37°C isotopes were added in 0.1 ml of 0.154 A4 NaCl and the incubation was continued for 120 min with air as the gas phase. In experiments with 5 &i of tritiated proline (TRA 82, The Radiochemical Centre, Amersham, England, batch no. 25, radiochemical purity > 98 %) as the precursor, the incubation medium contained 0.1 mM ascorbate and 14.5 ,uM L-proline, with 10 ,&i [3H]-D-glucosamine (TRK 375 batch no. 8, radiochemical purity 98 %) as the precursor 16.1 ,uM glucosamine hydrochloride, and with 2 ,&i [aH]cytidine (TRK 51 batch no. 11, radiochemical purity 98 %) as the precursor 0.33 ,uM cytidine. When [aH]cytidine or [3H]glucosamine was being incorporated, the incubation was stopped by cooling the samples to 0°C in an ice bath. Incubations with [3H]proline as the label were stopped by adding 270 pg of cycloheximide (Sigma) and 400 ,ug of a,a’-dipyridyl (Fluka) in 0.2 ml of incubation medium4. Aliquots were taken from the samples for cell counts with an electric cell counter (Celloscope 401, Ab Lars Ljungberg, Stockholm, Sweden) and for viability measurements with the Trypan-blue test 5. Average viability at the end of incubation was 70 % and was not dependent on the addition of serum or its fractions. No difference in this respect was found between test and control samples.

EFFECT OF HYPERLIPIDEMIC

After incubations

317

RAT SERUM ON SYNTHESIS OF COLLAGEN

the medium

was separated

from the cells by centrifuging

the

samples at 350 x g for 12 min and the cell pellet was washed once with the incubation medium, which in the [3H]proline incubations contained 0.01% cycloheximide and 0.0155% a,a’-dipyridyl. This washing supernate was discarded. The cells were suspended in 2.0 ml water and sonicated for 30 set (DiSONtegrator, System Forty, Ultrasonic Industries, Engineers’ Hill, L.I., New York, N.Y., U.S.A.). In the case of [aH]cytidine the medium and the cells were not separated, but 3.0 ml water was added to the incubated samples which were then sonicated. The cells and the medium fractions were dialyzed against cold running tap water for 24 hr, L-proline (0.05 mg/sample) or glucosamine hydrochloride (0.18 mg/ sample), respectively, being added as carriers. Analysis of the radioactive products. The dialyzates containing incorporated [aH]proline were hydrolyzed in 6 M HCl at 130°C for 3 hr. Hydrochloric acid was evaporated on a boiling water bath. Aliquots from the hydrolysates were taken for total radioactivity measurements and for the assay of [sH]hydroxyproline by the specific chemical procedure of Juva and ProckopG. The dialysates from the experiment with [sH]glucosamine were hydrolyzed in 2 M HCl at 103°C for 16 hr17. The hydrolysates were evaporated to dryness on a boiling water bath. The residue was dissolved in 2.0 ml water, and a 0.5 ml aliquot was taken for the radioactivity measurement. After the incorporation of [sH]cytidine, the dialyzates were hydrolyzed in 0.3 M KOH at 38°C for 16 hrs and one twentieth of the sample was taken for the radioactivity measurement. The radioactivities were measured with a Model 3320 Packard Liquid Scintillation

Spectrometer.

The counting

efficiencies

were determined

by using exter-

nal standardization. Production of hyperlipidemic rat sera Diets. White male Wistar rats aged 10-14 weeks, initial weight 273 i 6 g, were fed either a high-cholesterol olive oil diets (cholesterol concentration 15 g/kg; total calories, 38 % fat, 45 % carbohydrate, 17 % protein), a low-cholesterol olive oil diets (cholesterol cholesterol

concentration cocoa butter

0.03 g/kg, distribution of calories as before) or a highdiet10 (cholesterol concentration 50 g/kg; total calories : 68 % fat, 22 % carbohydrate, 10 % protein). The control rats received a standard laboratory diet for mice (Hankkija Oy, Helsinki, Finland; cholesterol concentration

0.4 g/kg, total calories: 10 % fat, 58 % carbohydrate, 32 % protein). In experiments where the effect of serum cholesterol on collagen synthesis was being studied the rats received one of the diets for 1-14 weeks ad Zibitum. Water was also given without restriction. The olive oil diets did not affect the weight gain of the rats, but the highcholesterol cocoa butter diet retarded the weight gain significantly, e.g., after 4 weeks the control rats were about 35 % heavier. Serum preparations. After 20 hr fasting, blood was collected by open heart puncture under light ether anesthesia and allowed to clot. The samples were centrifuged at 3000 x g for 15 min at room temperature to separate the sera. The density of 2-3 ml aliquots of sera was adjusted to 1.220 with NaCl-KBr-solution (0.154 M

318

T.

RijNNEMAA,

K. JUVA,

E. KULONEN

NaCI, d adjusted to 1.331 with potassium bromide) also containing 0.3 mM KzEDTAll, after which total lipoproteins were isolated by centrifugation at approximately 80,000 x g at 15°C for 40 hr with an MSE-65 ultracentrifuge equipped with a 6 x 15 ml swing-out density was increased

rotor (No. 59108). The top lipid layer was collected and its to 1.280 with the 1.331-NaCl-KBr solution. On top of this

sample two 2-ml layers of the NaCl-KBr solution, d 1.260 and 1.220 respectively, were carefully layered. The flotation centrifugation was repeated. The flotated lipid fraction and the original infranatant were dialyzed 3 times at 4°C against 2 1 0.154 M NaCl which contained 1 mM Kz-EDTA. This fractionation of the serum samples to lipoprotein and infranatant moieties was performed in those cases where a high activation of collagen synthesis by whole serum had been observed. This usually happened with rats fed high-cholesterol olive oil diet for 7 days (15 g/day). This feeding constantly induced hyperlipidemia with cholesterol concentration of about 110 mg/lOO ml. The lipid content of fractions was estimated by extracting the samples with chloroform-methanol (1: 1 by vol). After alkaline hydrolysis, cholesterol was determined from petroleum ether (boiling point 60-80°C) extract according to Badzio and Boczonlz and lipid-bound except that the digestion

phosphorus was determined as described by Bartlett13 was made according to Svanborg and Svennerholml4. and proteins according to Lowry Triglycerides were assayed according to Carlson15 et ~1.16,human serum albumin being used as standard.

??

5

.

z

=o * t-3

.”

.

.

z +

.

.

.

zroo-

. .

.

.

.

.

.. -

-

$

normal 0

hyperlipidcmlc

5era

I 0

, 200

100

Serum

cholesterol,

sew

300

mg/lOO

LOO

so0

ml

Fig. 1. Effect of cholesterol concentration of the added rat sera on the synthesis of collagen by embryonal fibroblasts (0). The rats received various diets for l-14 weeks. One-tenth ml serum was added to the incubation mixture. The mean of the collagen synthesis in experiments with sera from the control group is taken as 100.0. The mean for the group with serum cholesterol in the range of 100-150 mg/lOO ml (indicated by the dashed line), was 187 ‘A of the respective mean of the control group (solid linej.

EFFECT OF HYPERLIPIDEMIC

”

Serum

cholesterol,

OF COLLAGEN

LOO

mg/lOO

319

500

ml

performed as in Fig. 1, using the same sera but after dialysis (0).

I

TABLE EFFECT

300

Z””

I””

Fig. 2. Experiments

RAT SERUM ON SYNTHESIS

OF HYPERLIPIDEMIC

SERA

ON THE

SYNTHESIS

OF COLLAGEN

BY EMBRYONIC

FIBROBLASTS

Note: Hyperlipidemic (cholesterol concentration lW140 mg/lOO ml) or control sera were added to the incubation mixture in a volume of 0.1 ml. The volume of lipoprotein fractions added was adjusted to correspond to the amount of cholesterol in 0.1 ml of the original serum and the volume of the infranatants adjusted to correspond to the total protein content in 0.1 ml original serum. The values are based on 6 independent experiments, each including about 5 sera from both control and experimental rats. The mean incorporation in the control samples in each experiment was taken as 100%. Group of sera

Incorporation

of [3H]proline

control mean X!I S.E.M.

(n)

into collagen hyperlipidemic mean * S.E.M.

( %) P (n)

All samples Selected samplesa

100.0 + 6.9 (28) 100.0 & 7.4 (17)

142.8 & 12.1 (32) 171.9 f 14.0 (18)

< 0.01 < 0.001

Lipoprotein fractionb Infranatant fractionb

100.0 ??1.6 (17) 100.0 * 3.7 (17)

103.9 + 121.0 *

N.S. < 0.01

2.5 (18) 6.5 (18)

B Three most active sera in each experiment. b Density of the medium 1.220; selected samplesa. RESULTS

Eflect of serum cholesterol concentration on the activation of collagen synthesis The synthesis of collagen by embryonic fibroblasts increased with increasing cholesterol levels in the added sera up to a concentration of approximately 110 mg/lOO ml in whole serum but decreased with higher concentrations (Fig. 1). The cholesterol

320

T. RijNNEMAA, K. JUVA, E. KULONEN

TABLE 2 EFFECT

OF

EMBRYONIC

HYPERLIPIDEMIC

SERA

ON

THE

TOTAL

SYNTHESIS

OF

PROTEINS,

COLLAGEN

INCLUDED,

BY

FIBROBLASTS

Group of sera

Total incorporation control mean % S.E.M.

of [3H]proline

(n)

into protein

hyperlipidemic mean i_ S.E.M.

( y/o) P

(n)

All samples Selected sample+

100.0 & 4.5 (28) 100.0 f 4.0 (17)

123.9 + 8.6 (32) 128.7 + 9.4 (18)

< 0.05 < 0.01

Lipoprotein fraction” Infranatant fractionb

100.0 * 1.3 (17) 100.0 + 2.8 (17)

98.6 + 2.0 (18) 110.2 i 3.4 (18)

N.S. < 0.05

a Three most active sera in each experiment. b Density of the medium, 1.220; selected samples&.

concentration in the serum of control animals was about 45-55 mg/lOO ml. This stimulatory effect on collagen synthesis was dependent only on the serum cholesterol level and not on the diets or duration of feeding. Dialysis of the sera did not modify the effect (Fig. 2). The synthesis of non-collagenous proteins was not significantly affected by the varying cholesterol content. The average stimulatory activity of individual hyperlipidemic sera on collagen synthesis was 42.8 % (Table 1). A group of sera with an average stimulatory effect of 71.9 % was selected for further fractionation. In these sera the cholesterol concentration was 109.0 i 6.2 mg/lOO ml (49.5 & 2.5 mg/lOO ml in controls) and the phospholipid concentration

was 67.6 f

2.7 mg/lOO ml (76.2 f

3.3 mg/lOO ml in controls).

The stimulating agent was shown to stay in the infranatant fraction, while the total lipoprotein fraction was without effect. The cholesterol content in these infranatants of TABLE 3 EFFECT

OF HYPERLIPIDEMIC

Group of sera

SERA

ON THE

SECRETION

Collagen secretedc control mean i

S.E.M.

OF COLLAGEN

( %)

(n)

hyperlipidemic mean i S.E.M.

P (n)

All samples Selected samplesa

100.0 f 2.0 (23) 100.0 & 2.7 (14)

104.4 zt 2.3 (28) 108.1 zk 3.1 (15)

N.S. 0.10

Lipoprotein fractionb Infranatant fractionb

100.0 & 0.8 (14) 100.0 i 2.0 (14)

99.2 f 1.1 (15) 117.5 f 8.5 (15)

N.S. 0.10

& Three most active sera in each experiment. b Density of the medium, 1.220; selected samples”. r As the parameter to express the secretion of collagen, the ratio of the amount of radioactive hydroxyproline in the medium to the respective amount in the whole incubation mixture was calculated.

EFFECT OF HYPERLIPIDEMIC

TABLE EFFECT

321

RAT SERUM ON SYNTHESIS OF COLLAGEN

4

OF HYPERLIPIDEMIC

Two independent

SERA

ON

THEINCORPORATION OF [3H]~~~~~~~~~~~

experiments were made, each using 6-7 rats.

Group of sera

[3H]Clucosamine control mean * S. E.M.

incorporated

(n)

( %)

hyperlipidemic mean + S.E.M.

P (n)

All samples Selected samplesa

100.0 + 3.9 (12) 100.0 C 6.1 ( 7)

97.9 * 3.3 (14) 98.9 i 3.5 ( 7)

N.S. N.S.

Lipoprotein fraction h Infranatant fraction”

100.0 * 5.0 ( 7) 100.0 + 2.8 ( 7)

101.9 zl: 6.6 ( 7) 96.9 i 7.3 (7)

N.S. N.S.

& Three most active sera in each experiment. b Density of the medium, 1.220; selected samplesa.

TABLE 5 EFFECTOF HYPERLIPIDEMIC SERAON THEINCORPORATION OF [3H]CYTIDINE Group of sera

i3H]cytidine

incorporated

control mean & S.E.M.

(n)

( %) hyperlipidemic mean 31 S.E.M.

P (n)

All samples Selected sample@

100.0 + 4.7 (12) 100.0 t 8.2 ( 6)

101.8 f 9.4 (10) 106.6 f 14.8 ( 6)

N.S. N.S.

Lipoprotein fraction” Infranatant fractionb

100.0 + 0.7 ( 6) 100.0 i 2.4 ( 6)

103.7 i 105.1 +

N.S. N.S.

1.9 ( 6) 2.2 ( 6)

B Three most active sera in each experiment. b Density of the medium, 1.220; selected samplesa.

hyperlipidemic sera was around 1 mg/lOO ml, about twice the amount of that in the infranatants of control samples. A similar but smaller stimulatory effect on the total proline incorporation is shown in Table 2. Because about 70 % of the total protein synthesized is collagen, the results indicate that the synthesis of proteins other than collagen is increased very little, or even decreased. The effect of hyperlipidemia on the transport of collagen was evaluated by measuring the collagen excreted into the incubation medium. Some stimulating effect on collagen transport was demonstrated in the case of selected hyperlipidemic sera and their infranatants but not the isolated total lipoproteins (Table 3). Eflect of hyperlipidemia on the incorporation of glucosamine and of cytidine No effect was observed on the incorporation of either labelled glucosamine

or of

322 cytidine

T. RijNNEMAA,

(Tables 4 and 5). The stimulation

of collagen

K. JUVA, E. KULONEN

synthesis is not part of a general

connective tissue activation because there is no effect on the synthesis of acidic mucopolysaccharides or glycoproteins. Neither is the activation of collagen synthesis mediated

through

RNA.

DISCUSSION

The role of hyperlipidemia in the genesis of the changes associated with atherosclerosis has so far been studied mainly in vim, where it is hardly possible to detect the early events or the sequence of reactions appearing in individual cells. Besides, the effects of various serum fractions cannot be separated. The present experimental system contains only one cell type, which is rather well characterized. Its biological variation is small. The main protein synthesized and secreted into the medium is collagen4. The relevance of the information obtained with these highly specialized fibroblasts can be questioned because the main reacting cell in atherosclerosis

seems to be the arterial

smooth

muscle cell, which in the course of the

disease is known to proliferate and to produce extracellular matrix components including collagenl7-lg. Both the smooth muscle cells and tendon fibroblasts employed here are derived from mesenchymal tissue, which is always involved in regenerative processes, e.g., wound healing, hepatic cirrhosis, lung fibrosis and atherosclerosis. Most features associated with the biosynthesis and structure of collagen can be generalized to all the mesenchymal cells20although the various types of collagen differ in their primary structure and in some postribosomal modifications. The mechanism of the reactions

to exogenous

stimuli may still be similar.

Indeed,

the occurrence

of a

“myofibroblast” has been suggested to stress the analogy between the fibroblasts and smooth muscle ce1P. Because the eventual significance of the species difference between the test cells and the sera is not known, the results must be interpreted with caution in respect to the genesis of atherosclerosis. In this work the main emphasis was laid on the effect of hyperlipidemia on the biosynthesis and secretion of collagen and the comparisons were always made between test samples containing serum or its fraction from hyperlipidemic rat and control samples containing equal amounts of respective preparation from normolipidemic rat. Recently it has been shown that diet-induced hypercholesterolemia is associated granulation tissue of rat3. with increased collagen synthesis in vivo in experimental Our results are in agreement with that observation and further demonstrate that moderately hypercholesterolemic sera contain a factor that stimulates collagen synthesis in isolated fibroblasts. No increase in phospholipids in the sera was observed until the cholesterol concentration reached a level too high for the stimulation of collagen synthesis. Because the diets employed here did not induce hypertriglyceridemia, the stimulation of collagen synthesis observed is likely due to a factor associated with hypercholesterolemia. The stimulating principle was not dialyzable, suggesting its macromolecular

EFFECT OF HYPERLIPIDEMIC

RAT SERUM ON SYNTHESIS OF COLLAGEN

character.

that even though

cholesterol

It is remarkable content

the stimulating

of the serum, it was recovered

323

activity was related to the

in the infranatants

which contained

hardly detectable amounts of cholesterol, most of which is known to be bound to VHDL22. Ross et a1.l’ have shown that infranatant fraction of the serum of Macaca monkey either separately or combined with HDL and LDL preparations stimulates the proliferation of cultured aortic smooth muscle cells from the same species. However, their work does not deal with hyperlipidemia and it lacks information on the effect of lipoproteins

without

infranatant

fraction,

thus leaving

the question

on the

location of the stimulating factor in the serum somewhat open. The appearance of the stimulating factor in the infranatant fraction in the serum of rats developing hyperlipidemia suggests the possibility that the stimulation of fibroblasts by lipids occurs through an unknown mediator. This is in accordance with the finding of Heppleston et aL23, that silica irritates fibroblasts to overproduction of collagen through a mediating substance excreted by macrophages. There seem to exist several connective tissue activating factors, for example, those introduced recently by Castor et al.24 and by McGee et a1.25. Especially relevant here is the finding that a fibroblast-stimulating peptide can be isolated from carbon tetrachloride-damaged mouse liver 25. The question arises whether the stimulating factor reported in the present paper is also formed in the liver.

REFERENCES 1 CONSTANTINIDES, P., Experimental Atherosclerosis, Elsevier, Amsterdam, New York, London, 1965, pp. 14-24. 2 FULLER, G. C., MILLER, E., FARBER,T. AND VANLOON,E., Aortic connective tissue changes in miniature pigs fed a lipid-rich diet, Conrz. Tissue Res., 1 (1972) 217. 3 PELLINIEMI,T.-T., Lipids of Connective Tissue, Thesis, Turku, 1973. 4 DEHM, P. AND PROCKOP,D. J., Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon, Biochim. Biophys. Acta, 240 (1971) 358. 5 MERCHANT,D. J., KAHN, R. H. AND MURPHY, N. H., Handbook of Cell and Organ Culture, Burgess, Minneapolis, Minn., 1964, p. 157. 6 JUVA, K. AND PROCKOP,D. J., Modified procedure for the assay of 3H- or lW-labelled hydroxyproline, Anal. Biochern., 15 (1966) 77. 7 LEHTONEN,A., The mucopolysaccharides in ageing experimental granulation tissue, Acta Physiol. &and., Suppl. 310 (1968) 25. 8 RANTANEN,J., Radiation injury of connective tissue, Acta Radio/., Suppl. 330 (1973) 32. 9 SAITO,S. AND FILLIOS,L. C., Effects of dietary lipids on hepatic protein synthesis and lipid metabolism in the rat, Amer. J. Physiol., 208 (1965) 882. 10 RENAUD,S., ALLARD,C. AND LATOUR,J. G., Influence of the type of dietary saturated fatty acid on lipemia, coagulation and the production of thrombosis in the rat, J. Nutr., 90 (1966) 433. 11 HAVEL, R. J., EDER, H. A. AND BRAGDON,J. H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 12 BADZIO,T. AND BOCZON,H., The determination of free and esterified cholesterol in blood after separation by thin layer chromatography, Clin. Chim. Acfa, 13 (1966) 794. 13 BARTLETT,G. R., Phosphorus assay in column chromatography, J. Biol. Chern., 234 (1959) 466. 14 SVANBORG,A. AND SVENNERHOLM, L., Plasma total lipid, cholesterol, triglycerides, phospholipids and free fatty acids in a healthy Scandinavian population, Acta Med. Stand., 169 (1961) 43. 15 CARLSON,L. A., Determination of serum triglycerides, J. Atheroscler. Res., 3 (1963) 334. 16 LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chew., 193 (1951) 265,

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T. RijNNEMAA, K. JUVA, E. KULONEN

17 Ross, R. AND GLOMSEX, J. A., Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 18 CAVALLERO, C., TONDO, U., MINGAZZINI, P., BARONI, C., PESANDO,P. AND CAVALLERO,M., Smooth muscle cell proliferation in the reaction of the arterial wall to injury. In: Proceedings of Carlo Erba Foundation, Milan, 1973, pp. 3-8. the International Symposium on Arteriosclerosis, 19 GEER, J. C. AND HAUST, M. D., Smooth muscle cells in atherosclerosis. In: Monographs on Atherosclerosis, Vol. 2, Karger, Basle, 1972, pp. 19-26. 20 GRANT, M. E. AND PROCKOP,D. J., The biosynthesis of collagen, New Engl. J. Med., 286 (1972) 194. 21 GABBIANI,G., MAJNO, G. AND RYAN, G. B., The fibroblast as a contractile cell - The myo-iibroblast. In: E. KULONENAND J. PIKKARAINEN (Eds.), Biology of Fibroblast, Academic Press, London, New York, 1973, pp. 139-154. 22 SCANU, A. M., Structural studies on serum lipoproteins, Biochint. Biophys. Acta, 265 (1972) 471. 23 HEPPLESTON,A. G. AND STYLES,J. A., Activity of a macrophage factor in collagen formation by silica, Nature, 214 (1967) 521. 24 CASTOR,C. W., SMITH,S. F., RITCHIE,J. C. AND SCOTT,M. E., Connective tissue activation Mechanism and significance. In: E. KULONENAND J. PIKKARAINEN(Ed%), Biology of Fibroblast, Academic Press, London, New York, 1973, pp. 483499. 25 MCGEE, J. O’D., O’HARE, R. P. AND PATRICK, R. S., Stimulation of the collagen biosynthetic pathway by factors isolated from experimentally injured liver, Nature New Biol., 243 (1973) 121.