Sulfated glycosaminoglycans synthesized by fibroblast, smooth muscle and endothelium-like cells grown in culture

Cell Differentiation, 12 ( 1983) 99-108 Elsevier Scientific Publishers Ireland, Ltd.

99

Sulfated glycosaminoglycans synthesized by fibroblast, smooth muscle and endothelium-like cells grown in culture P a u l o A.S. M o u r ~ o ~, S r e e k u m a r Pillai 2 and Patricia V. D o n n e l l y 2 I Departamento de Bioquimica, Centro de CiOncias da Sahde. Universidade Federal do Rio de Janeiro, 21941. Rio de Janeiro. R J. Brazil," and 2 Department of Biochernist£v. B~(vlor College of Medicine, Houston. TX. U.S.A. (Accepted 3 June 1982)

Monolayer cultures of fibroblast, smooth muscle and endothelium-like cells incorporated 35SO~ into glycosaminoglycans of the extracellular, pericellular and intracellular compartments. These glycosaminoglycans have been identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The sulfated glycosaminoglycans from the extracellular pool of the three cell types show a similar composition, while the intracellular and pericellular pools of the three cells have a different glycosaminoglycans composition. They differ in the relative proportion of heparitin sulfate and chondroitin sulfate and in the structure of isomeric chondroitin sulfate. glycosaminoglycans

fibroblasts

smooth muscle

endothelium-like cells

1. Introduction

Evidence for the biosynthesis of sulfated glycosaminoglycans by fibroblasts, smooth muscle cells and endothelium cells in culture has been established (Buonassisi, 1973; Kresse et al., 1975; Wight and Ross, 1975; Conrad et al., 1977; Blake and Conrad, 1979; Middendorf et al., 1980; Namiki et al., 1980). These polymers can be isolated from the culture media (extracellular pool) and from the cell layer. By mild trypsinization of the cells, part Abbreviations used: GlcUA ~ GalNAc4S, 2-acetamido-2-deoxy3-0(/~-D-glucopyranosyluronic acid)-4-O-sulfo-D-galactose (disaccharide type A); GIcUA ~ GalNAc6S, 2-acetamido-2-deoxy-3-O-(/~-D-glycopyranosyluronic acid)-6-O-sulfo-D-galacrose (disaccharide type C); I d U A ~ G a l N A c 4 S , 2-acetamido2-deoxy-3- O-( a-L-idupyranosyluronic acid)-4- O-sulfo-D-galacrose (disaccharide type B). Chondroitin 4,6-sulfate and dermatan sulfate are also known as chondroitin sulfate A / C and chondroitin sulfate B, respectively. Chondroitin sulfate is used to designate copolymers of chondroitin 4,6-sulfate and dermatan sulfate. PPO, 2,5-diphenyloxazole; CETAVLON, Ncetyl-N,N,N-trimethylammonium bromide; ECTEOLA, epichlorohydrin triethanolamine

monolayer cultures

of the glycosaminoglycans can be removed and is usually referred to as 'membrane associated' or 'pericellular pool' (Kraemer, 1971). The 'intracellular pool' is the glycosaminoglycans that remain in the cells after mild trypsinization. The presence of heparitin sulfate at the external surface of cells has been reported for a number of different cell types grown in vitro (Dietrich and DeOca, 1970; Kraemer, 1971; Kraemer and Tobey, 1972; Chiarugi et al., 1974). Chondroitin 4,6-sulfate and dermatan sulfate are also present in the cellular and extracellular compartments of cells grown in vitro (Conrad et al., 1977; Dietrich and DeOca, 1978; Mourfio and Machado-Santelli, 1978; Blake and Conrad, 1979; Mourfio et al., 1980). In the present study we have prepared sulfated glycosaminoglycans from the pericellular, intracellular and extracellular compartments of fibroblast, smooth muscle and endothelium-like cells. The relative proportion of different sulfated glycosaminoglycans and the polymeric structure of the chondroitin sulfates are reported. It was observed that the extracellular pool of the three cell

0045-6039/83/0000-0000/$03.00 ~9 1983 Elsevier Scientific Publishers Ireland, Ltd.

100 types shows a similar sulfated glycosaminoglycan composition, while the pericellular and intracellular pools of the different cell types differ from each other.

2. Materials and Methods

35S - L a b e l e d C e l l C u l t u r e s

Nedia

Ceils

I

Dialysed against 0.15 M NaCl

Washed t w i c e w i t h phosphate-buffered

gCIEO~ Ceilulose c o l u ~ chromatography

2 ml of 0.05% trypsin

I1

2.1. Cell cultures The smooth muscle and endothelium-like cells derived from bovine aorta were kindly supplied by Dr. S.G. Eskin (Baylor College of Medicine, TX, U.S.A.), while the fibroblast-like cells were derived from human skin biopsy. Previously, microscopic studies revealed that these cells have the structure characteristics of smooth muscle cells, endothelium cells and fibroblasts, respectively (Kruse and Patterson, 1973). Further characterization of the cultures of endothelium and smooth muscle-like cells used in this study has been reported previously (Eskin et al., 1978, 1980). Confluent monolayer cultures were prepared in 60 X 15 m m petri dishes in 2 ml of Eagle's minimal essential medium (EMEM) (Gibco) containing 10% fetal calf serum (FCS) (Paul, 1970: Kruse and Patterson, 1973). Dishes were incubated at 37°C in humid chambers in an atmosphere of 5% CO2/95% air. The medium was changed twice per week. The experiments were performed when the cultures reached confluence.

2.2. Labeling of cells When the cultures were confluent monolayers, H~5SO4 (30 ~Ci/dish) was added in fresh nutrient media containing 10% FCS. The cultures were maintained in the continuous presence of radioactive media for 72 h. Previous studies indicate that within a 72 h incubation period a constant amount of [~SS]glycosaminoglycans is found in the pericellular and intracellular pool, while the amount of [-~SS]glycosaminoglycans in the media grows linearly with time for the duration of the experiment (Namiki et al., 1980: Middendorf et al., 1980).

2.3. Isolation of the glycosaminoglycans The procedures for isolation of ~SS-labeled glycosaminoglycans from the extraceIlular, pericellu-

i n 0 . 0 5 % I. . . . . . . 1 0 m i n

Fr~¢tlon eluted with 4.0 M NaCI

200D x g f o r ~ i n

-~&

...... distilled water

Supe~atant

(:ell pellet

Vacuum d r i e d

2 ml o f 0 . 0 5 2 EDTA f o r

....... trypsin

0.0,

li

....... .... Ppt with 4 volumes of 95% ethanol a t -I09C for 24 b

. . Ppt w i t h 4 v o l u m e s of 95% ethanol at -IO?C for 24 h

Dissolved distilled

D i s s o l v e d i n 100 ~l dlsti]led water

ill 10O ~1 water

EXTP.ACELLULAR GLYCOSAMINOGLYCANS

12 h

a t 37~C

II

in 0.05g

PERICELLULAR GLYCOS~MINOGLYCANS

.

.

.

. Ppt with 4 volumes of 95% ethanol at -IOQC for 24 h

Dissolved distilled

i n 100 ~i water

I NTI~ACELhULAR GLYCOSAMINOC[,YCANS

Fig. 1. Procedures for the isolation of the 35S-sulfated glycosaminogtycans (GAG) from the intracellular, pericellular and extracellular fractions of the cultured cells. lar and intracellular fraction of the cultured cells are briefly illustrated in Fig. 1. At the completion of the labeling period, the media were decanted and the cells were rinsed with phosphate-buffered saline (PBS), pH 7.4. The cells were detached by trypsinization with 2 ml of 0.05% trypsin in 0.05% EDTA for 10 rain at room temperature. The trypsinate was centrifuged (2000 X g for 10 rain at room temperature) and the supernatant, representing the pericellular glycosaminoglycans, was separated from the cell pellet, whereafter it was resuspended in 2 ml 0.05% trypsin containing 0.05% E D T A and incubated overnight at 37°C. The trypsin-digested cell pellet and the pericellular fraction were then brought to pH 11.0 with 0.1 N N a O H and left for 8h at room temperature. These solutions were neutralized with 0.1 N HC1 and the glycosaminoglycans precipitated with 4 volumes of 95% ethanol; maintained at - 1 0 ° C for 24h. The precipitate formed was collected by centrifugation (5000 X g for 15 min at

101

10°C), washed once with 80% ethanol and vacuum dried. The dried material was dissolved in 100 ~1 of distilled water. For studies of the glycosaminoglycans from the extracellular fraction, the media were dialyzed at 4°C against 0.15 M NaCI. The proteoglycans from the media were then isolated by E C T E O L A cellulose column chromatography (Nakashima et al., 1975), as follows: E C T E O L A cellulose (OH form) was equilibrated with 0.15 M NaC1 and packed in a column 10 × 1 cm. The media were applied to the column, which was then washed with 0.15 M NaC1 until the washings were free of 35SO4 radioactivity. The column was then eluted with 10 ml of 4.0 M NaCI. The proteoglycans eluted from the column were exhaustively dialyzed against distilled water at 4°C and vacuum dried. The dried material was dissolved in 1.0 ml of 0.05% trypsin in 0.05% E D T A for 12h at 37°C. After incubation, the glycosaminoglycans in the supernatant solution were precipitated with 4 volumes of 95% ethanol and maintained at - 1 0 ° C for 24h. The precipitate formed was collected by centrifugation (5000 X g for 15 min at 10°C), washed once with 80% ethanol and vacuum dried. The dried material was dissolved in 100/xl of distilled water. To eliminate the possibility of monospecific trapping of the 3s - S O 2~ in the culture media~ as well as to check for possible cell-free synthesis, parallel flasks containing culture media without cells were labeled and the media were extracted for glycosaminoglycans as described above. To estimate the amounts of -~sS-labeled glycosaminoglycans present in each one of the different pools (extracellular, pericellular and intracellular), after the 72h labeling period, 25 p.1 aliquots of each solution were applied to Whatman No. 1 chromatography paper and developed in isobutyric a c i d / 1 N N H 3 (5:3, v / v ) for 24 h (Mour~o et al., 1980). The material that remained in the origin of the chromatograms was quantitated in 10 ml 0.5% P P O / t o l u e n e solution in an L-S Beckman spectrometer.

2.4. Identification of the ~3S-labeled glycosaminoglycans For the identification of the 35S-labeled glycosaminoglycans two criteria were used: electro-

phoretic mobilities in agarose gel and the enzymatic degradation with chondroitinase AC and ABC. The agarose gel electrophoresis was carried out as previously described (Dietrich and Dietrich, 1976) in 0.05 M 1,3-diaminopropane: acetate buffer pH 9.0. The -~-sS-labeled glycosaminoglycans prepared from the cells were visualized by radioautography of the stained gels, using Kodak XO m a t X-ray film, exposed for 5 days (Mourfio et al., 1980). The radioautograms were quantitated by densitometry using a Quick Scan densitometer (Helena Laboratories, Beaumont, TX). Chondroitin 4-sulfate (Sigma Chemical Co., St. Louis, MO), chondroitin 6-sulfate (Miles Laboratories, Elkhart, IN), heparitin sulfate, and dermatan sulfate, kindly supplied by Dr. J.A. Cinfonelli (University of Chicago, IL), were used as standards. Enzymatic degradation of the glycosaminoglycans was performed by incubation of 30/xl of each solution (about 10,000 c.p.m.) with 0.01 units of chondroitinase A C I I (Arthrobacter aurescens) or ABC (Proteus vulgaris) (Miles Laboratories, Elkhart, IN) in 0.05 M ethylenediamine: acetate buffer p H 8.0. After 12h at 37°C, the mixtures were applied to Whatman No. 1 chromatography paper and developed in isobutyric a c i d / l N NH.~ (5 : 3, v / v ) for 24 h. The strip of the chromatogram containing the products formed from standard glycosaminoglycans was stained with silver nitrate (Mour~o et al., 1973), while the remaining part was used for radioautography, as already described. The radioactive bands with identical chromatographic migration of the standard disaccharides were cut and quantitated in 10 ml 0.5% P P O / t o l u e n e solution in an L-S Beckman spectrometer. The amounts of disaccharide unit types A, B or C were estimated after d e g r a d a t i o n with chondroitinases AC and ABC, as described by Saito et al. (1968). The final products of these enzymes are unsaturated disaccharides, namely AGlcUA-~ GalNAc4S, 2-acetamido-2-deoxy-3-O(/~-D-glyco-4-enepyranosyluronic acid)-4-O-sulfoD-galactose, and A G l c U A ~ G a l N A c 6 S , 2acetamido-2-deoxy-3-O-(/~-D-glyco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose. The amounts of 4- and 6-sulfated unsaturated disaccharides

102

f o r m e d b y c h o n d r o i t i n a s e A C e n a b l e us to estim a t e the a m o u n t s of r e p e a t i n g d i s a c c h a r i d e units c o n t a i n i n g glucuronic acid in the c h o n d r o i t i n sulfate molecule, m a i n l y G l c U A - ~ G a l N A c 4 S (A units) a n d G l c U A ~ G a l N A c 6 S (C units). As the u n s a t u r a t e d d i s a c c h a r i d e f o r m e d from d e r m a t a n sulfate a n d c h o n d r o i t i n 4-sulfate by the action of the c h o n d r o i t i n a s e A B C is the same, n a m e l y AGlcUA---, G a l N A c 4 S , and the d e r m a t a n sulfate is resistant to the action of the c h o n d r o i t i n a s e A C , the difference between the a m o u n t s of A G l c U A ~ G a l N A c 4 S f o r m e d by c h o n d r o i t i n a s e A C or A B C enables us to estimate the a m o u n t s of r e p e a t i n g d i s a c c h a r i d e units c o n t a i n i n g i d u r o n i c acid in the c h o n d r o i t i n sulfate molecule ( I d U A -~ G a l N A c 4 S , B units). The r a d i o a c t i v i t y m a t e r i a l that r e m a i n e d in the origin of the c h r o m a t o g r a m s after the action of c h o n d r o i t i n a s e A B C indicates the labeled heparitin sulfate in the mixture. In some e x p e r i m e n t s this m a t e r i a l was eluted with water, c o n c e n t r a t e d to 10 /~1 a n d a n a l y s e d by agarose gel electrophoresis. O n l y one b a n d , m i g r a t i n g as h e p a r i t i n sulfate, was detected.

3. Results

3.1. Distribution of the -¢sS-labeled glycosaminoglycans in the intracellular, pericellular and extracel/ular fractions T a b l e I shows the total and relative a m o u n t s of the 35S-labeled g l y c o s a m i n o g l y c a n s in the intracellular, pericellular and extracellular pools of fibroblast, s m o o t h muscle a n d e n d o t h e l i u m - l i k e cells after 72 h i n c u b a t i o n period. All cell types i n c o r p o r a t e d 35S-sulfate into the glycosaminoglycans of the three fractions, but c o n s i d e r a b l e variations in the total a n d relative a m o u n t s of the 35S-labeled g l y c o s a m i n o g l y c a n s were observed. 35S-Sulfated g l y c o s a m i n o g l y c a n s are in a p p r o x i m a t e l y equal a m o u n t s in the intracellular a n d pericellular fractions of fibroblasts; in e n d o t h e l i u m - l i k e cells, most of the [35S-]glycosaminoglycans from the cellular layer are in the intracellular pool, whereas for the s m o o t h muscle-like cells app r o x i m a t e l y 84% of the [35S]glycosaminoglycans p r e s e n t in the cell layer are in the pericellular fraction a n d only 16% are in the intracellular

TABLE I Distribution of 35S-labeled glycosaminoglycans in the intracellular, pericellular and extracellular fractions of different cell types Cell type

Fibroblast-like cells Smooth muscle-like cells Endothelium-like cells

Fraction

Intracellular Pericellular Extracellular Intracellular Pericellular Extracellular lntracellular Pericellular Extracellular

~sS-Labeled glycosaminoglycans c.p.m./10 ~ cells

~

21,305 23,905 87,615 21,412 l 16,401 82,610 293,562 154,164 296,400

16 18 66 10 53 37 39 21 40

Fibroblast, smooth muscle and endothelium-like cells were labeled by 72 h incubation with 35SO42 (30 ktCi/dish). The radioactivity of the glycosaminoglycans present in the intracellular, pericellular and extracellular compartments was measured and expressed as c.p.m./plate.

103

compartment. Fibroblast-like cells secreted 66% of the [35S]glycosaminoglycans synthesized in the 72 h labeling period, whereas smooth muscle-like cells secreted only 37% and endothelium-like cells 40%.

and heparitin sulfate. Those obtained from the pericellular fraction show bands corresponding to heparitin sulfate and chondroitin 4,6-sulfate, while the extracellular fraction shows a major band that migrates between dermatan sulfate and chondroitin 4,6-sulfate, and a less intense one corresponding to heparitin sulfate. The densitometry of the electrophoresis and the relative proportions of the various 35S-sulfated glycosaminoglycans from these three compartments are indicated in Fig. 2B. The 3-sS-labeled glycosaminogly~ans were also analysed by enzymatic degradation with chondroitinase AC and ABC. The degradation products formed were analysed by paper chromatography and located in the chromatogram by radioautography. The quantitative results are presented in Table II. Only trace amounts of 35S-

3.2. Identification of the -~sS-labeled glycosaminoglycans obtained from the three compartments q[ fibroblast-like cells The glycosaminoglycans from intracellular, pericellular and extracellular pools of fibroblastlike cells were extracted and analysed by agarose gel electrophoresis. The radioautogram of the agarose gel electrophoresis of the 35S-labeled glycosaminoglycans obtained from these compartments is shown in Fig. 2A. The 35S-sulfated glycosaminoglycans from the intracellular fraction show two bands that migrate as dermatan sulfate

A

40,

B

~

a , , - - * ~ ,~;,~,,~ ....

20

b • ~ .......

" /''

~ cs ~-DS ~ --H$

__~-'~'~ __ ,

ACELLULAR

~

F

--ORI6IN

s'-;

201 ~ O~

i'"

/ ~

~/,," '

,,/' ~0 ,I" HS

DS

ELECTROPHORETIC

,;

'

~/

,~

INTRACELLULAR

'

5'0

CS MIGRATION

(MM)

Fig. 2. Agarose gel electrophoresis of the 35S-labeled glycosaminoglycans produced by fibroblast-like cells grown in culture. A) About 2000 c.p.m, of the 35S-labeled glycosaminoglycans from the intracellular (I), pericellular (P) and extracellular (E) compartments of fibroblast-like cells grown in culture were submitted to agarose gel electrophoresis (0.05 M 1,3-diaminopropane/acetatepH 9.0 for 1 h at 120 V). The glycosaminoglycans in the gel were fixed with C E T A V L O N and stained with 0.1% solution of toluidine blue in acetic a c i d / e t h a n o l / w a t e r (0.1:5:5). The radioactive bands corresponding to the 35S-sulfated glycosaminoglycans were detected by radioautography of the electrophoresis, performed with Kodak X-Omat X-ray film, exposed for 5 days. St, standard mixture containing 10 /~g each of chondroitin 4,6-sulfate (CS), dermatan sulfate (DS) and heparitin sulfate (HS). a) Radioautography; b) Toluidine blue staining. B) Densitometry of the electrophoretic radioautograms using a Quick Scan densitometer (Helena Laboratories). The broken lines indicate the electrophoretic migration of standard chondroitin 4,6-sulfate (CS), dermatan sulfate (DS) and heparitin sulfate (HS). The numbers inside the electrophoretic bands indicate the relative amounts of the various 35S-labeled glycosaminoglycans.

104 TABLE II Products formed by chondroitinases AC and ABC upon the 35S-labeledglycosam~noglycansfrom different fractions of fibroblast-like cells •

Enzyme

Products

,,

Cellular fraction Intracellular

Pericellular

Extrucellular

c.p.m.

%

c.p.m.

%

c.p.m.

9;

Chondroitinase AC

non-degraded (origin) AGIcUA~ GalNAc4S &GIcUA~ GalNAc6S

20,967 1,598 790

90 7 3

8,511 2.086 1.327

71 18 11

4.801 2,176 1,067

60 27 13

Chondroitinase ABC

non-degraded (origin) AGIcUA~ GalNAc4S AGIcUA ~ GalNAc6S

15,336 6,030 995

69 27 4

7,742 3,511 1,728

60 27 13

4,190 3,912 1,208

45 42 13

3sS-Labeled glycosaminoglycansobtained from different compartments of fibroblast-like cells were incubated with 0.01 units of chondroitinase AC and ABC for 13 h at 37°C in 0.05 M ethylenediamine:acetatc buffer, pH 8.0 in a final volume of 40 #1. After incubation, the mixture was applied to Whatman No. 1 paper and subjected to chromatography in isobutyric acid/1 N NH~ (5:3, v/v) for 24 h. The products formed were located in the chromatogram by radioautography. The radioactive bands with the same chromatographic migration of the standard disaccharides were cut and quantitated in 10 ml 0.5% PPO/toluene solution in a L-S I00 Beckman spectrometer.

labeled oligosaccharides or 3~S-disulfated disaccharides are formed by c h o n d r o i t i n a s e ABC ( < 5% of total products). C h o n d r o i t i n sulfate from intracellular fraction is composed of a p p r o x i m a t e l y 23% type A, 14% type C and 63% type B disaccharide units; that from the pericellular fraction is composed of 33% type A, 40% type C a n d 27% type B disaccharide units, while that from extracellular fraction c o n t a i n s 42% type A, 24% type C a n d 34% type B disaccharide units. As the c h o n d r o i t i n sulfates from different c o m p a r t m e n t s show a single electrophoretic b a n d , analysis of the results o b t a i n e d in Table II suggests that these c h o n d r o i t i n sulfates of fibroblast-like cells have a hybrid structure composed of three types of disaccharide units.

3.3. Identification of the -~sS-labeled glycosarninoglycans obtained from the three compartments of smooth muscle-like cells T h e a g a r o s e gel e l e c t r o p h o r e s i s of the -~sS-labeled glycosaminoglycans o b t a i n e d from the three c o m p a r t m e n t s of smooth muscle-like cells is shown in Fig. 3. The -~-SS-sulfated glycosaminoglycans from the intracellular a n d pericellular frac-

tions show b a n d s corresponding to c h o n d r o i t i n 4,6-sulfate a n d heparitin sulfate. The intracellular fraction c o n t a i n s mainly heparitin sulfate (62%), while the pericellular fraction c o n t a i n s mainly c h o n d r o i t i n 4,6-sulfate (63%). Sulfated glycosa m i n o g l y c a n s from extracellular fraction show a major b a n d that migrates between d e r m a t a n sulfate a n d c h o n d r o i t i n 4,6-sulfate a n d another, less intense, c o r r e s p o n d i n g to heparitin sulfate. The degradation products formed by the action of c h o n d r o i t i n a s e A C a n d ABC u p o n the )sS-labeled glycosaminoglycans are shown in Table III. Again, only trace a m o u n t s of 35S-labeled oligosaccharides are formed by chondroitinase ABC ( < 5 % ) . The results presented in Table III indicate that c h o n d r o i t i n sulfate from the intracellular pool is composed of approximately 74% type A and 26% type C disaccharide units a n d that from the pericellular pool is composed of 60% type A a n d 40% type C disaccharide units. Only m i n o r a m o u n t s of type B disaccharide units ( < 5%) are detected in these two pools. C h o n d r o i t i n sulfate from the extracellular pool is composed of approximately 49% type A, 29% type C and 22% type B disaccharide units.

105

A

B

~

a r

b

~

~

20

m ----_

~

RACELLULAR

z :~

¸lC$ ~l-DS

0

°

P

E

~"

.

.

.

.

.ol.

~

I

~

A, 0~" ~, - A,, ,, : ' 20~ J~.~NT,,O,kh.h,,

~l-H$

~

,

--ORIGIN

.

o~.,;

~

,'~o

HS

,' ,

DS

(

.'~o

CS

ELECTROPHORETIC

it

~

"

;0

MIGRATION

.

(MM)

Fig. 3. Agarose gel electrophoresis of the 35S-labeled glycosaminoglycans produced by smooth muscle-like cells grown in culture. The glycosaminoglycans extracted from the various compartments of smooth muscle-like cells grown in culture were analysed by agarose gel electrophoresis as described in Fig. 2.

TABLE IIl Products formed by chondroitinases AC and ABC upon the ~sS-labeled glycosaminoglycans from different fractions of smooth muscle-like cells Enzyme

Products

Cellular fraction |ntracellular

Pericellular

Extracellular

c.p.m.

%

c.p.m.

%

c.p.m.

%

Chondroitinase AC

non-degraded (origin) AGlcUA ~ GaINAc4S AGIcUA ~ GalNAc6S

3,003 1,243 414

64 27 9

11,812 9,970 6,647

42 35 23

11,060 4,761 2,886

59 26 15

Chondroitinase ABC

non-degraded (origin) AGIcUA ~ GalNAc4S AGlcUA ~ GalNAc6S

2,949 1,284 428

63 28 9

12,503 10,511 6,415

42 36 22

9,846 6,871 2,736

51 35 14

Experiments were performed as described in Table lI.

3.4. Identification of the ~sS-labeled glycosaminoglycans obtained from the three compartments of endothelium-like cells Fig. 4 shows the agarose gel electrophoresis of the [3-~S]glycosaminoglycans extracted from the in-

tracellular, pericellular and extracellular compartments of endothelium-like cells. The intracellular and pericellular fractions show a predominating band migrating as heparitin sulfate, while the extracellular fraction shows a major band (71%) that migrates between dermatan sulfate and chondroi-

106

A

B

21! EXTRAOELL 4O

b r

~

~

~-CS

40 I-

I-DS

E

I-HS

t~l

•

T

~

s~

~"

/

// /

7:

~ 4O ¢-~

(~

/

A

20

o I~,

/

--ORIGIN

20 0 20

,' 30 HS

40 DS

ELECTROPHORETIC

50

CS MIGRATION

(MM)

Fig. 4. Agarose gel electrophoresis of the 35S-labeled glycosaminoglycans produced by endothelium-like cells grown in culture. 3sS-Labeled glycosaminoglycans extracted from the various compartments of endothelium-like cells grown in culture were analysed by agarose gel electrophoresis as described in Fig. 2.

for the other cellular types, only minor amounts ( < 5%) of 35S-disulfated disaccharides or [3_SS]oligosaccharides are formed by chondroitinase A B e . The combined data from Fig. 4 and Table IV suggest the presence of hybrid chondroitin sulfate in the three compartments and allow esti-

tin 4,6-sulfate, and a less intense one (23%) corresponding to heparitin sulfate. The [35S]glycosaminoglycans extracted from the three compartments of endothelium-like cells were degraded with chondroitinases AC and ABe. The disaccharides formed are indicated in Table IV. As

T A B L E IV Products formed by chondroitinases AC and A B e upon the 35S-labeled glycosaminoglycans from different fractions of endothelium-likc cells Enzyme

Products

Cell fraction lntracellular

Pericellular

Extracellular

c.p.m.

%

c.p.m.

%

c.p.m.

%

Chondroitinase AC

non-degraded (origin) AGIcUAv/GalNAc4S AGIcUAcGalNAc6S

50,650 1,748 1,946

93 3 4

21.815 1,273 1.652

88 5 7

2,768 422 607

73 11 16

Chondroitinase A B e

non-degraded (origin) AGIcUAr, GalNAc4S AGlcUA¢'GalNAc6S

50,462 2,580 1,567

92 5 3

20.038 1,702 1,493

86 7 7

2,220 650 603

64 19 17

Experiments were performed as described in Table 11.

107

mation of the relative proportion of type A, B and C disaccharide units. Chondroitin sulfate from the intracellular fraction contains 42% type A, 38% type C and 20% type B disaccharide units; that from the pericellular fraction contains 40% type A~ 47% type C and 13% type B disaccharide units, while that from extracellular fraction contains 45% type A, 44% type C and 11% type B disaccharide units.

4. Discussion

The question of whether different cells contain or secrete different types of sulfated glycosaminoglycans was recently approached by several authors. Sulfated glycosaminoglycans which differ in their structure, molecular weight and relative proportion were isolated from various organs (Toledo and Dietrich, 1977). However, since the sulfated glycosaminoglycans were produced by a mixture of different cell types present in these organs, that question remained unanswered. The results described in this paper have demonstrated that fibroblast, smooth muscle and endothelium-like cells grown in vitro synthesized sulfated glycosaminoglycans, which were, in part, secreted to the media. While the extracellular pool of the three cell types shows a similar sulfated glycosaminoglycans composition, the pericellular and intracellular pools of the different cell types differ from each other. The total [3-SS]glycosaminoglycans are in almost equal amounts in the pericellular and intracellular pools of fibroblasts, while in smooth muscle-like cells they are in a higher concentration in the pericellular pool and in the endothelium-like cells in the intracellular pool. Heparitin sulfate and chondroitin sulfate are present in different proportion at both compartments of the three cell types. However~ the endothelium-like cells are distinguishable by the high proportion of heparin sulfate in pericellular and intracellular fractions. Chondroitin sulfate with a high proportion of iduronic residues (type B disaccharide units) is present mainly in the intracellular pool of fibroblast-like cells and absent in the intracellular and pericellular pools of smooth muscle-like cells.

Chondroitin 4,6-sulfate is the predominating 3-~S-sulfated glycosaminoglycans in the pericellular pool of fibroblast and smooth muscle-like cells. The extracellular pool of the fibroblast, smooth muscle and endothelium-like cells contains heparitin sulfate and isomeric chondroitin sulfate composed of type A, B and C disaccharide units. Higher concentrations of heparitin sulfate in the extracellular pool were obtained by the chondroitinase method (chondroitinase ABC-resistant [~-SS]glycosaminoglycans) than by agarose gel electrophoresis. Such results could be explained by the presence of low molecular weight heparitin sulfate, which would not precipitate by CETAVLON in the agarose gel after the electrophoresis. The synthesis of macromolecules and their secretion to the media by cells grown in culture have been extensively studied. It was shown that some substances added to the culture media interfere with the secretion of glycosaminoglycans (Kurtz and Stidworthy, 1975; Nakamura et al., 1980), and might be simulating the influence of the interstitial matrix composition on the metabolism of the connective tissue. Also, changes in some of the original properties of cells, in relation to the synthesis and secretion of macromolecules, have been reported when the cells are grown in vitro. Mayne et al. (1977), for example, have shown that synthesis of a high proportion of type III collagen by guinea pig aortic smooth muscle cells was only observed in the first passage of these cells from the initial outgrowth. For subsequent passages, the cells showed a more "fibroblastoid' behavior with less type III than type 1 collagen, although they retain the morphological characteristics of smooth muscle cells. The results presented here showing that different cell populations secrete to the media [~SS]glycosaminoglycans with a similar composition may be a consequence of changes in some of the original properties of the cells caused by the conditions of culture in vitro. Another possibility is that the culture media control the amount and structure of the sulfated glycosaminoglycans synthesized by the cells and secreted to the media. In this case, since fibroblast, smooth muscle and endothelium-like cells were grown in the same culture media, the type and relative proportion of the secreted

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sulfated glycosaminoglycans would be expected to be the same. Such possibility raises an interesting question concerning the interference of the interstitial space composition of the connective tissue in the synthesis and secretion of sulfated glycosaminoglycans by the various cell populations. For these reasons it is difficult to extrapolate the data obtained by the study of the sulfate glycosaminoglycans produced by the cells grown in culture to those produced in vivo. Nevertheless, some interesting correlations might be drawn: the skin and the aorta adventitia layer, dermatan sulfate-rich tissue (Toledo and Dietrich, 1977: Toledo and Mourn,o, 1979), are also rich in fibroblasts, while the aorta intima layer, where the endothelium cells are the main cell type, also contains higher amounts of heparitin sulfate when compared to the media and adventitia layer (Toledo and Mourgto, 1979).

Acknowledgements This research was aided by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), Financiadora de Estudos e Projetos ( F I N E P - B / 7 6 / 7 9 / 0 8 2 ) , Fundaq~,o de Amparo fi Pesquisa do Estado de Silo Paulo (FAPESP-80/1847-1) and by an N I H / F o g a r t y International Research Fellowship (TW 02856-02). The authors wish to express their gratitude to Dr. Y.M. Michelacci for reading the manuscript.

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