The effect of heparin on fibronectin and thrombospondin synthesis by human smooth muscle cells

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1264-1269

November 13, 1987

THE EFFECT OF HEPARIN ON FIBRONECTIN AND THROMBOSPONDIN SYNTHESlS BY HUMAN SMOOTH MUSCLE CELLS

Bernadette Lyons-Giordano, Helen Conaway, and Nicholas A. Kefalides Departments of Medicine and Biochemistry and Biophysics and the Connective Tissue Research I n s t i t u t e University of Pennsylvania and the University City Science Center Philadelphia, PA 19104 Received September 25, 1987

Heparin causes increased synthesis of fibronectin and thrombospondin by human vascular smooth muscle c e l l s as assessed by immunoprecipitation and ELISA techniques. More fibronectin and thrombospondin were immunoprecipitated from the medium of c e l l s treated with 180 ~g/ml heparin than from that of control c e l l s . Heparin did not effect levels of f i bronecti n and thrombo spondi n immunopreci pi tated from the cel I -matrix fractions. By ELISA, heparin was found to cause a 1.7 fold increase in medium fibronectin l e v e l s / c e l l and a I0 fold increase in medium thrombospondin levels/cell. Concomitantly, smooth muscle c e l l s treated witn 180 g/~11 neparin for 48 h exhibited 55% decrease in p r o l i f e r a t i o n r e l a t i v e to controls. SUMMARY:

©

1987 Academic Press, Inc.

Numerous studies angiogenesis.

In

have implicated vivo

studies

heparin

using

rat

as an important carotid

arteries

modulator

of

injured

by

denudation of the endothelium have shown heparin to be a potent i n h i b i t o r vascular smooth muscle cell in

suppressing

anticoagulant

(SMC) p r o l i f e r a t i o n

SMC p r o l i f e r a t i o n (I).

The

is

inhibitory

(I).

independent effects

of

of

The efficacy of neparin of

its

heparin

function on

as

vascular

an SMC

p r o l i f e r a t i o n have been confirmed in v i t r o using rat SMC (2). Heparin

enhances

the

proliferation

of

human endothelial

cells

(3).

Previously, we nave reported that heparin causes a specific and dose dependent decrease in fibronectin

(FN) synthesis without affecting

synthesis by human endothelial FN synthesis

was related

human endothelial heparin

inhibits

cells

c e l l s (4).

closely (4,5).

human vascular

to In

the this

thrombospondin (TSP)

The i n h i b i t o r y effect of heparin on heparin-enhanced report,

SMC p r o l i f e r a t i o n

proliferation

we provide

evidence

of that

and causes a concomitant

increase in both FN and TSP synthesis by these c e l l s .

Abbreviations: SMC, Smooth muscle cells; FN, fibronectin; TSP, thrombospondin; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; T r i s , tris(hydroxymetnyl) amino methane; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme linked immunosorbent assay. 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

1264

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

CELL CULTURE: Preparation of SMC from human umbilical cord arteries was conducted e s s e n t i a l l y as described by Tumilowicz (6). Cells were grown in Dulbecco's modified Eagles medium with 10% fetal c a l f serum, 1 mM glutamine, 15 mM HEPES, 0.4 ~g/ml amphotericin B and I0 pg/ml gentamicin in the presence and absence of 180 ~g/ml heparin (Sigma, St. Louis, Missouri). Cells were maintained at 37°C in a humidified 5% CO? atmosphere. METABOLIC LABELING: After washing the c e l l s twice with phosphate buffered saline, the subconfluent cultures were incubated for 24 h at 37oc with 8 uCi/m] [35S] methionine (Trans [35S]-label, ]090 Ci/mmole, IC~J Radiochemicals, Irvine, CA) in methionine-free Dulbecco's modified Eagles medium supplemented witn 1 mM glutamine, 0.1% glucose, 50 ~g/ml ascorbic acid, 50 ~g/ml B-aminoproprionitrile fumarate, 15 mM HEPES, and in the presence and absence of ]80 ug/ml heparin. For analysis, the medium was collected and protease inhibitors, N-ethylmaleimide (I mM) and phenylmethylsulfonylfluoride (ImM), were added. To rule out possible effects of heparin during ti~e analytical procedures, heparin was added to medium from control cultures at the termination of the incubation to a final concentration of 180 ~g/ml. Cell debris was sedimented by centrifugation at 500 x g for 20 min at 4°C. Cell-matrix layers were extracted with 62.5 mM Tris-HC| (pH 6.8) containing 2% SDS and 4M urea. SDS-PAGE: Proteins in the medium and c e l l - m a t r i x samples were analyzed by SDS-PAGE according to Laemmli (7) using 5% resolving gels. Prior to analysis, medium samples were dialyzed against O.5M acetic acid and l y o p h i l i z e d . The samples from the medium and c e l l - m a t r i x fractions were dissolved in 62.5 mM Tris-HCl (pH 6.8) containing 4 M urea, ~% SDS, 10% glycerol, 0.0025% bromophenol blue and 5% 2-mercaptoethanol. Gels w e r e prepared for Fluorography using En3Hance (NEN, Boston, Mass.), and after drying, were exposed to X-O-Mat ×AR-5 film at -70oc. [14C]-labeled FN, isolated from the medium of [14C]-proline labeled human endothelial cells, was used as a standard (8). IH~4UNOPRECIPITATION: Radiolabeled FN and TSP were iiT~nunoprecipitated from the medium and c e l l - m a t r i x fractions recovered a f t e r metabolic labeling of cultures in the absence and presence of heparin (180 ~ g / m l ) . The immunoprecipitation was conducted using protein A sepharose beads as described by Bumol and Reisfeld (9). Rabbit antibody to human FN and FN standard were purchased from Biomedical Technologies (Stoughton, MA) and rabbit antibody to I1~Jman TSP and TSP standard were g i f t s from Dr. George Tuszynski, Lankenau Hedical Research Center, L a n k e n a u Hospital, Philadelphia, PA. Immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. ELISA: For Quantitation of FN and TSP in the medium of heparin-treated and control human SMC, cultures were incubated in serum-free medium (as described For metabolic labeling conditions) for 24 h at 37°C. ELISA was performed as described by Rennard et al (lO). CELL PROLIFERATION ASSAY: Human SMC were seeded at a density of 1.5 x I04 cells/cm 2 at passages from 3-8. For heparin-treated cultures, heparin (180 ~g/ml) was present in the growth medium from 12 h to 60 h after seeding. After 60 h, the cells were trypsinized and counted using a Coulter counter. Net growth was calculated by subtracting the number of cells seeded from the number of cells recovered by trypsinization.

RESULTS The effects studied

of

heparin on human SMC biosynthesis

using metabolic labeling and ELISA techniques.

1265

of TSP and FN were Cells,

pre-treated

Vol. 148, No. 3, 1987

1

2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

4

5 i

2

3

5

4

6

7

8

9

i0

11

12

FN-FN I

TSP" TSP--

Q)

(i) FIGURE I . The e f f e c t of heparin on FN and TSP synthesis by human SMC. SMC were pre-lncubated in growth medium containing 180 ug/ml heparin for 24 h at 37oc and then labeled in the presence of heparin (180 pg/ml) for 24 n at 37oc with [35S]methionine. Control SMC were pre-incubated and labeled in the absence of heparin. Radiolabeled proteins recovered in the medium and c e l l - m a t r i x fractions of the heparin-treated and control cultures were fractionated by SDS-PAGE and visualized by autoradiography. Lane I , FN standard; lane 2, medium from control SMC; lane 3, medium from heparin-treated SMC; lane 4, c e l l - m a t r i x fraction from control SMC; lane 5, c e l l - m a t r i x fraction from heparin-treated SMC. FIGURE 2. Immunoprecipitation of FN and TSP from the medium and c e l l - m a t r i x fractions of heparin-treated and control SMC. TSP and FN in the medium and c e l l - m a t r i x fractions from heparin-treated (180 pg/ml) and control SMC labeled for 24 h at 37°C with [35S]methionine were analyzed by immunoprecipitation. Lanes 1 and 2, immunoprecipitates of medium from control and heparin-treated SMC, respectively, using antibody to TSP; Lanes 3 and 4, immunoprecipitates of c e l l - m a t r i x fractions from control and heparin-treated SMC, respectively, using antibody to TSP; lanes 5 and 6, immunoprecipitates of medium from control and heparin-treated SMC, respectively, using pre-immune serum; lanes 7 and 8, immunoprecipitates from c e l l - m a t r i x fractions of control and heparin-treated SMC, respectively, using pre-immune serum; lanes 9 and I0, immunoprecipitates of medium from control and heparin-treated SMC, respectively, using antibody to FN; lanes I I and 12, immunoprecipitates of c e l l - m a t r i x fractions from control and heparin-treated SMC, respectively, using antibody to FN.

with

heparin

for

24 h,

m e t h i o n i n e and h e p a r i n . absence of h e p a r i n . levels

were l a b e l e d Control

the

of both FN and TSP i n the medium.

tion

proteins.

using r a b b i t

indicating

These data were f u r t h e r antibodies

specific

24 h w i t h

medium c o n t a i n i n g

were p r e - i n c u b a t e d

As shown i n Figure I ,

FN and TSP l e v e l s were observed, of

for

cells

for

1266

and l a b e l e d

[35S] in

the

heparin caused an increase i n the No e f f e c t

on c e l l - m a t r i x

no e f f e c t

substantiated TSP or FN.

associated

on c o m p a r t m e n t a l i z a t i o n by immmunoprecipita-

As shown i n F i g u r e 2,

Vol. 148, No. 3, 1 9 8 7

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I: ELISA analysis of the effect of neparin on human smooth muscle cell synthesis of FN and TSP ug FN/IO6 cells Control

2.22

Heparin-Treated

3.81

~g TSP/IO6 cells 1.21 12.4

Heparin-treated human SMC were preincubated 24 h at 37oc in growth medium containing 180~g/ml heparin. The cultures were then incubated 24 h at 37o in serum-free medium containing 180 ~g/ml heparin. At the termination of the incubation, the medium was collected for ELISA and the cells were trypsinized and counted. Control cultures were pre-incubated in growth medium and incubated in the absence of heparin. Medium levels of FN and TSP auantitated by ELISA were normalized by cell number for comparison between control and heparin- treated cultures.

tnore FN was immunoprecipitated from the medium of heparin-treated c e l l s from the medium of control c e l l s . of

Little

FN was evident between control

dramatic

increase

treated c e l l s . fractions.

in

and heparin-treated c e l l s .

TSP immunoprecipitated

(FN is

there

from

the

There was a

medium of

heparin-

No TSP was detected in the i i ~ u n o p r e c i p i t a t e s from c e l l - m a t r i x apparent

in

i~lunoprecipitates

treated samples due to non-specific Since

than

d i f f e r e n c e in the c e l l - m a t r i x l e v e l s

is

little

or

binding

the medium f r a c t i o n s ,

medium from

heparin-treated

the

the

cell-matrix

greater l e v e l s

human SMC must

normal

rabbit

serum

of FN to protein A sepharose.)

no FN and TSP in

compared to

of

reflect

fractions

as

of TSP and FN in

the

augmented synthesis

of

these proteins. To Quantify the e f f e c t human

SMC, E L I S A analyses

heparin-treated ('180 ~g/ml) 37°C

of beparin on on the synthesis of FN and TSP by

in

serum-free

were

conducted

and control

medium.

on

medium c o l l e c t e d

cultures a f t e r

Heparin-treated

cells

from

a 24 n incubation at

were

pre-incubated

for

24 11 in heparin (180 ~g/ml) supplemented growth medium. Heparin induced a 1.7 f o l d increase in medium FN l e v e l s / l O 5 c e l l s and a I0 f o l d increase in medium TSP

levels/lO6

cells

(Table

i).

T n e s e data

are

consistent

with

the

metabolic l a b e l i n g r e s u l t s . The e f f e c t of heparin on human SM~ growth i s treated f o r

48 h with 180 ~g/ml

r e l a t i v e to control

cultures.

shown in Table I I .

heparin e x h i b i t e d 55~ i n h i b i t i o n

Thus,

heparin i s

a potent i n h i b i t o r

Cells

of

growth

of human

SMC p r o l i f e r a t i o n .

DISCUSSION In t h i s paper, we have shown t h a t heparin i n h i b i t s human SMC p r o l i f e r a t i o n and augments synthesis of TSP and FN by these c e l l s .

The a n t i - p r o l i f e r a t i v e

e f f e c t s of heparin on SMC in c u l t u r e had been demonstrated using r a t a o r t i c SMC ( I ) .

It

has p r e v i o u s l y been reported t h a t heparin a l t e r s the secretory 1267

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

TABLE II:

The effect of heparin on human smooth muscle cell growth

NET GROWTH

% INHIBITION OF GROWTH

Control

5.94xi05 +/- 0.29xi05

Heparin-Treated

2,68xi05 +/- O.18xlO5

55%

Human SMC were seeded at 1.5xlO 4 cells/cm 2. 12 h after seeding, cells were fed with growth medium with or without 180 P g/ml heparin and allowed to proliferate for 48 n. Cells were then trypsinized and counted. Net growth is the difference between the number of cells seeded and the number of cells recovered. Values given are averages of triplicate determinations +/standard deviation. phenotype of vascular SMC ( I I , 1 2 ) . demonstrated

that

non-collagenous

heparin

selectively

polypeptides

expression of

type III

Using rat a o r t i c SMC, Majack and Bornstein

(Mr=37,000

and type

artery

a o r t i c SMC; r a t h e r ,

(ll).

It will

the

synthesis

39,000)

and

as well

of

t~o

modulated

the

as that of a short

In contrast to our Findings with

SMC, heparin did not a l t e r

TSP synthesis

in

the rat

increased TSP levels in the medium induced by heparin were

associated with decreased incorporation (12).

and

I procollagens

chain (Mr=60,O00) collagenous protein human umbilical

induced

of TSP into the e x t r a c e l l u l a r

matrix

be i n t e r e s t i n g to learn whether t h i s apparent difference in the

e f f e c t of heparin on TSP r e f l e c t s a species or tissue s p e c i f i c i t y

or n e i t h e r .

The e f f e c t s of heparin on human vascular SMC growth and biosynthesis of FN and TSP are the inverse of i t s Heparin,

in

endothelial with

a

the

presence

cell

endothelial

of

proliferation

specific

decrease

cells

(4,5).

e f f e c t s on human vascular endothelial endothelial (3).

in

cell

growth

This p r o l i f e r a t i v e

FN

synthesis,

but

factor,

effect

not

TSP

Thus, in both human endothelial

of

this

relationship

however,

since

TSP

is

not

the

synthesis

same f o r

is

also

SMC as

modulated

for

enhances

is associated synthesis,

cells

synthesis is inversely related to the rate of p r o l i f e r a t i o n .

by

and SMC, FN

The s p e c i f i c i t y

endothelial

with

cells.

cells;

changes

in

SMC

proliferation. An abundance of l i t e r a t u r e

has h i g h l i g h t e d the importance of e x t r a c e l l u l a r

matrix molecules and cytoskeletal and

differentiation

(for

decreases FN synthesis 3H tnymidine endothelial

components in the regulation of cell see 14).

Our

in human endothelial

cells

incorporation

review (15)

suggests

that

observation prior

that

growth neparin

to the induction

decreased

FN synthesis

c e l l s may be a p r e r e q u i s i t e f o r stimulation of growth.

of by

Similarly,

decreased FN synthesis also may be permissive for human SMC growth in as much as p r o l i f e r a t i n g

SMC synthesize less FN t n a t g r o w t h - i n h i b i t e d SMC. Decreased

medium l e v e l s

of

linkages

thereby

endothelial

and

cells.

FN may r e s u l t provide

in

altered

growth

The physiologic

substratum-receptor-cytoskeletal

stimulatory

relevance of 1268

signal(s)

to

SMC and

heparin as a modulator

of

Vol. 148, No. 3, 1987 vascular cell

growth is

BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS suggested by the finding that endothelial cells

p~oduce heparan sulfate moieties with heparin-like growth inhibitory functions for SMC (16),

ACKNOWLEDGEMENTS The authors thank Ms. Maryann Mason for typing the manuscript. was supported by NIH grants AR-20553 and HL-29492.

This ~ork

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