Synthesis of interstitial collagens by pig aortic endothelial cells in culture

Vol. 84, No. 3, 1978 October

BIOCHEMICAL

RESEARCH COMMUNICATIONS

16, 1978

SYNTHESIS

OF

Pages

INTERSTITIAL M.J.

Medical

AND BIdPHYSICAL

Research University

Received

Barnes,

14,

BY PIG

L.F.

Council, Cambridge,

of

August

COLLAGENS

Morton

Connective Tennis

AORTIC and

ENDOTHELIAL

C.I.

646-653

CELLS

IN

CULTURE

Levene

Tissue Team, Department Court Road, Cambridge,

of U.K.

Pathology,

1978

SUMMARY: Examination of the collagens synthesized by pig aortic endothelial min culture and precipitated from either the cell layer or medium, following pepsin digestion, demonstrated that the major species was Type I together with some Type III collagen and al (I) trimer. Additional chains in the cell layer chromatographing with Type I al-chains on CM cellulose may be partly derived from basement-membrane-associated collagen but in the medium would appear to be entirely derived from al (I) trimer. These results imply that the endothelial cell may secrete the Type III collagen located in the immediate subendothelial space and, in part at least, the Types I and III occurring in diffusely thickened intima and the atherosclerotic plaque. Endothelial described of

the

offers

cells

as

of

basement

first

synthesize,

permit

collagen

that

endothelial Types

definitive

the has

space I and

diffusely

III

in

primarily

the

this

have

cell

medial

wall been

(authors'

Collagen the

cultured

may

and

detected

by

smooth

muscle

I and

at

has cell

Type

immediate least

data)

sites

can III>.

the

chemical

been

been

derivation

cells

the

(or

has

communication

secrete in

these

the

(Types

unpublished at

with

endothelial

type

(4)

that

This

collagens

vessel

intima

(5).

accord

immunohistochemically

that

human

in

(1,2,3)

endothelium.

that

that

blood

collagens

from

the

interstitial

located

plaque

from

evidence the

been

collagen

(1,2)

inference

thickened

atherosclerotic originate

synthesize

membrane

additionally,

findings

culture

basement-membrane-type

vascular the

in

in

III sub-

part)

analysis and

Gur

in

considered

the in

the to

(6).

MATERIALS AND METHODS: Culture of endothelial cells Endothelial cells were obtained as before (3) from pig aorta by the method of Jaffe et al (7) as modified Endothelial cells so isolated and then subcultured 5-6 by Leake and Bowyer (8). times in 199 medium containing 20% foetal calf serum (FCS), were kindly provided by the latter authors. For the purposes of the present investigation, cells were grown in 60 mm Falcon dishes at an initial density of 50,000 in 4 ml of Dulbecco and Vogt's modification of Eagle's bIEM containing 10% FCS. Medium was exchanged for fresh every three days. After six days, ascorbic acid (50 u,g/ml) was added to the medium. At specified days, indicated later, medium was replaced with fresh containing either L- [G-3H] proline or L- [4,5-3H ]lysine (generally 0.5 mCi/dish) and ascorbic acid (50ug/ml). After 24h, plates were either 0006-291x/78/0843-0646$01.00/0

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

646

Vol. 84, No. 3, 1978

BIOCHEMICAL

AND BIdPHYSICAL

RESEARCH COMMUNICATIONS

harvested or a further supplement of ascorbic acid was given. In the latter case dishes were harvested 24h later. Radiolabelled collagens in the cell layer and medium were examined as described below. Examination of cell layer collagens Digestion of cell layers, dispersed in 0.5M acetic acid, with pepsin, precipitation of the digest at 0.9M NaCl and the separation of precipitated collagens by differential salt precipitation, all in the presence of carrier were undertaken as before (9). Fractions precipitating at I, 1.71, 2.1 and 2.56M NaCl were collected and examined by CM cellulose chromatography. Examination of collagens in the medium The medium following dialysis was digested with pepsin as described previously (9). The digest was dialysed against 0.41 Na phosphate buffer, pH 7.6. After addition of carrier collagen (prepared by precipitation of a pepsin digest of pig aorta by dialysis against 0.02M Na2HP04) at approx 3mg/ml, collagens in the digest were precipitated by dialysis against 0.02M phosphate. The precipitate was redissolved in the 0.41 buffer and then incubated overnight at 37O (IO) to effect the thermal gelation of interstitial collagens. The gel was centrifuged for 30 min at 10,000 g at 25O. The residue (containing interstitial collagens) and supernatant were examined by CM cellulose chromatography. A portion of the supernatant was subjected to a second gelation by means of a modification of the above procedure (Dr. J. Rauterberg, unpublished data). The sample after addition of carrier collagen at approx 3 mg/ml was diluted to 0.21 , incubated at 37O for 6h and then dialyzed at 20° against IO vol H 0 overnight. The precipitate was collected by centrifugation as above. CM cellu f ose chromatography was exactly as before (9). Radioactivity in the eluate was measured as before (II) except that PCS (Amersham/Searle Corp.) diluted 2:1 (v/v) with xylene, was used as scintillant. Gel chromatography was undertaken with a column (140 x 1.5cm) of Sepharose CL-6B; conditions were otherwise as before (9). Radioactivity in the eluate was measured as above. Collagenase digestion Samples of the cell layer, after dispersal (IO), or medium or fractions derived therefrom were dialysed against 0.05M tris-HCl, pH7.g (at 25O) containing O.OiM Ca acetate and 0.02% Na azide, denatured at 100 for 5 min, and then redialyzed. Collagenase (Worthington's CLSPA purified and treated as described by Benya et al (12))was added at 20 units/ml. Samples were incubated for 6h at 370, then dialyzed against water and the diffusate in each case lyophilized prior to hydrolysis. Hydrolysis Samples were heated in 6N HCl in sealed tubes at 1080 for 24h. Hydrolysates were dried in vacua in a dessicator over cont. H2S04 in the presence of NaOH pellets. Measurement of lysine and hydroxylysine radioactivity in hydrolysates was as before (II). Measurement of proline and hydroxyproline radioactivity in hydrolysates.proline and hydroxyproline were separated on a Technicon Amino Acid Autoanalyzer at 60° using the standard citrate buffer, pH 2.9. Radioactivity in the eluate was counted as above. RESULTS:

In initial

studies

collagenase-susceptible the period

indicated

the % conversion

material

of lysine

in cultures

labelled

to hydroxylysine [ 3 H] lysine

with

in during

was as follows:-

Expt.

I;

5-7d:

Cell

layer,

19; Medium,

Expt.

2; 6-7d:

Cell

layer,

5; Medium,

Since

basement-membrane

(Type

24. 44.

IV) collagen

647

IO-12d:

Cell

layer,

26; Medium,

23.

12-13d:

Cell

layer,

15; Medium,

52.

exhibits

values

of about

80% (13-17)

BIOCHEMICAL

Vol. 84, No. 3, 1978

the

figures

above

This

was

C3~]

proline-labelled

taken

by

a more

confirmed

Examination

was

in

pro/hyp

that

collagenase was

upon (of

than In

one Expt.

activity as

salt

collagens

2:i

and

the

in

Na2

HP04

there The

obtained

not a ratio

obviously appreciably

material

was

the % distribution collagens

pepsin

collagenous

certainly,was

This

the

(18).

respectively)

(where

of

separate

remainder,

unprecipitated

collagen

lo-lad),

two in

Of

0.02M 4:l

the and,

fractionation

synthesized

In

NaCl.

against

(13-17)).

from

collagens.

hydroxyproline

O.QM

dialysis

anticipated

1 (labelled

following

of

examined

total

precipitated

no

further.

radio-

at

O.QM

NaCl

was

follows:-

II,! ppt,

4; In

1.71~

and

al:a2:

ratio al(I)

known

1)

revealed

al

(I)

and

of

5 in

region,

usually

16;

with

(Fig.

fraction

the

ppt,

accord

cellulose

at

with

to

precipitate

have

been

shown Expt.

M ppt,

the a2

2.56

a higher Type

generally

III

collagen

from

Type

I in

the

2.56M

indicated

presence

either

molarity

than

(24))

or

an of

perhaps

vicinity

of

Q-lld),

the

in

NaCl

of (al) (19-23)

IV

the

1.711 The

chains

in

trimer

(which

collagen a-chains

(I)

chains

% distribution

CM

but

and al

21.

fraction.

I

Type

(25-27)

on

excess

Type

2.56M

2M NaCl the

- soluble,

chromatography

Type

in

from

2.56M

of

around

chromatograph

2 (labelled

I

34;

presence

fraction the

M ppt,

bebaviour,

chains

latter

normal

to

25;

precipitation

suggesting

known

In

2.1

the

precipitates

coprecipitate

as

be

interstitial

layer

["II]

at

IV)

the

cell

total

material (Type

might

the

suggested

under-hydroxylated

basement-membrane-related less

in

approx

treatment

of

below.

precipitated

ratio

of

described

the

RESEARCH COMMUNICATIONS

synthesis

analysis

of

precipitation

following moiety

as

two-thirds

radioactivity

the

collagens

material

negligible

indicate

detailed

radio-labelled approx.

digest

to

cultures

of

experiments,

was

were

AND BidPHYSICAL

can which

from

in is

which

(13).

of

radioactivity

2.56

M-soluble,

was

follows:-

1M PPt,

9;

In

instance

this

cellulose

1.71

M ppt, , most

chromatography

17;

2.1

M ppt,

of

the

radioactivity

again

54;

indicated

2.56

4;

precipitated the

648

M ppt,

presence

at of

Type

2.lM III

part

NaCl. collagen

16. CM:

Vol. 84, No. 3, 1978

BIOCHEMICAL

0

Figure

10

the

1.7iM

located The

M

I 70

60

in

al(I)

elevated

al:a2

suggesting

rather was

a single since

close

be

in

Type

gel a-chain

the

in

was

CM cellulose

of

of

and

the

the

position

The of

precipitated

the 2.561

al

al-peak

revealed

21,

Type

pro/hyp

Type

IV

radioactivity

fraction

revealed (not

I,

a

perhaps to

(I)-chains

after

2).

was

attributable

However peaks.

(Fig.

a2-peak

(Fig.

were

was

I collagen The

greater

al(I)

these

that

Type

However,

size

ppt.

radioactivity

noted,

(14-17,281.

in

material

again again

regionof

all

fraction,

filtration.

I trimer

unity

represents

nature

pro/hyp

trimer. the

on

of

5)

2.lM

I).

is

only

shown)

and

considered

al(I)-trimer. The

The

to

peak this

chains

the

indicative

by

peaks

than

In

(approx. size

with

2.56M

shown).

regions

ratio

excess

collagen

~2

a-chain

pattern

ratio

(not and

of

complex

1.71M ppt.

fraction

essentially

to

M 40 FRACTION NUMBER

RESEARCH COMMUNICATIONS

1. Endothelial cells in culture: CM cellulose chromatography of the precipitates at 1.71 and 2.561 NaCl obtained in the fractionation of radiolabelled cell layer collagens isolated after pepsin treatment (Expt. Badioactivityina 0.5 ml aliquot of each 10 ml fraction of the eluate was counted in 10 ml of scintillant at 30% efficiency. The position of marker chains aa indicated was established by chromatography of appropriate unlabelled collagens. The results relate to the cell layers pooled from three plates, each labelled with 0.3 mCi[L G-3H] proline from lo-12d.

-----------

in

al

AND BIdPHYSICAL

of

derived

collagenous

radioactivity However

front

the

and from

ratio

Cbl cellulose failed

Type

material

to

reveal

V collagen

of

1.4

soluble

was

compatible

chromatography either which

al could

(18,24.29,30).

649

at

with

exhibited (I)

chains

also

in

2.56M

only or

theory

USA! occur

NaCl its

is

uncertain.

identity

a single and in

as peak

~1: Bl this

al at

chains fraction

(I)

Vol. 84, No. 3, 1978

BIOCHEMICAL

2.(A) CR cellulose chromatography of the precipitate at 2.1M NaCl obtained in the fractionation of radiolabelled cell layer collagens isolated after pepsin treatment (Expt. 2). Radioactivity in a 0.5 ml aliquot of each 10 ml fraction of the eluate was counted in 5 ml of scintillant (in polypropylene inserts) at 20% efficiency. The results relate to the cell layers pooled from six plates, each labelled with 0.5 mCi L [G3H] proline from g-lid; one third of the total sample was chromatographed. (B) Gel filtration of al(I) chains: Fractions 22-31 of the above chromatogram were pooled, dialyzed against 0.15M acetic acid, lyophilized and a portion then applied to a column of Sepharose CL-6B. Radioactivity in a 0.5 ml aliquot of each 2.5 ml fraction of the eluate was counted as in Fig. 1. The position of elution of chains as indicated was established by gel filtration of appropriate unlabelled samples.

Examination

of radiolabelled

of interstitial subjected basement

types, to thermal

membrane

dialysis

collagens

gelation derived

against

species

0.02M

Na2HPO4.

to reflect

possibly

degradation

(31,32).

A pro/hyp

radioactivity

appeared

to preclude

appropriate

fraction

0.02M

the

(10)

its

phosphate

ratio

only

of the yielded

in Methods.

collagenous

from

turnover

a residue

650

the

that

front

2) were from 25% of by

failed

to

and was considered of collagen

collagenase

IV collagen.

fraction

Expt

precipitated

(16)

a peak at the

synthesis

Approx.

30% NaCl

of 1.5 following

from Type

(from

75%) of the remainder

arising

the

of interstitial

the fraction

even with

products

derivation

revealed

gelation

(about

carrier)

medium

separation

as described

Most

To confirm

in the

in the medium was in

(with

The initial

collagens

to effect

precipitate

precipitated

in the medium.

radiolabelled

the C3H ] hydroxyproline

against

RESEARCH COMMUNICATIONS

a2 1

T al(l)

Figure

AND BIdPHYSICAL

treatment

The 30% NaClon CM cellulose.

precipitated

represented

by dialysis

approximately

40%

Vol. 84, No. 3, 1978

BIOCHEMICAL

AND BIdPHYSICAL

a2

RESEARCH COMMUNICATIONS

y(m)

3A

Y

a

I

I

II

I

20 1-

I

I

0

LO

Figure

a.2

1

a FRACTICN NlJMI)ER

40

\[:1,,

,

5u

0

M

,

..i m

83

PO

,

,-

\

im 110 120 ELUIAR VOtUNT Imll

lx)

la

3. (A) CM cellulose chromatography of the radiolabelled collagens from the medium (Expt 2) precipitated by gclation at 37O: One quarter of the The eluate was counted as in Fig. I. (B) Fractions total sample was applied. dialyzed and lyophilized and 44-57 of the above chromatogram were pooled, The eluate was counted as in applied to a column of Sepharose CL-6B. The y-chains are presumed to be derived from the Type III peak Fig. 1. and the a-chains from the Type I a2 peak.

of the total

radioactivity

in

revealed

the presence

Type III

peak was of y-chain

as Type III approx

2:l

collagen

(Fig.

chains.

precipitate

only that

about

gelation

of interstitial

collagens)

radioactivity

in the

and a2-chains

with

4 suggested

a major

yielded

supernatantl amount al-chains

their

carrier in the

was found

it

found it

supernatant.

to seemed

Application over

about contained

thought

Type I

in the vicinity

90%

75% of the mostly

The al(I): is

of from

collagen,

(representing

of Type III-chains.

651

derivation

to precipitate

by CM cellulose

and must

identification

in a ratio

was subsequently

a fraction that

were

The

its

peak on C?d cellulose

Type I must remain

excess

with

technique

(which

3).

confirming

of the unlabelled

procedure

chromatography (Fig.

and a2 peaks

consistent

the gelation

a small

collagens

filtration

The al(I)

revealed

some labelled

CM cellulose

I and III

by gel

size

a half

of the modified

of about

size

of a-chain

Since

fraction.

Types

3).

The supernatant

of al(I)

likely

of both

and were

collagen.

this

reflect

U2 ratio the

al(I)

Vol. 84, No. 3, 1978

presence only

of al(I)

about

trimer.

5% of the

chromatography

The final

initial

The cells

not

only

of recognisable

used

in view appearance

as monolayers

at confluence.

character.

cells.

by the

latter

(at

and this

would

It

we observed

muscle

cells.

is not

possible

as opposed

of collagen

arising

through

chondrocytes collagen phase

synthesis later

The synthesis

(34) its

no simple producing

synthesis

arises the rate is

smooth

whether

distinction

(Type

V collagen)

chick

cornea1

epithelium

are

labelled

phase

as well has been

of interstitial

a change

for

in the

character example

a similar

with

pattern

of

"endothelial"

cells

cells

in culture

of epithelial

type

(IV and V) and the fibroblast

interstitial

collagens

shown to synthesize

as Type shown

of contaminating

in the early

between

collagens

have been

cells

collagens

synthesis

by endothelial

can be drawn

cells

by endothelial

(3).

collagens

producing

We find

(33).

with

of C3H]proline

of phenotypic

as can occur

all)

of our cultures

represents

of loss

cells

the cells

of interstitial

chains

collageens

not in

interstitial

the

from

interstitial

a low percentage

whether

as a result

'fibroblastic'

muscle

from

at present

muscle

is

in

remained

much (if

than

that

also

they

of incorporation

much lower

endothelial

but

by contamination

of the cells,

or smooth

forms

in culture

be derived

basement-membrane

differentiated

Similarly

cells

the possibility

to state

contained

on CM cellulose

employed

that

could

gelation

entirely

demonstrate

our hands)

sub-culturing (19)

or the

implies

of isolation

to basement-membrane-related

pattern

considered

Our studies

seem to preclude

levels

were

since

this

second

and revealed

importantly

by these

in

the

3) and most

In any event, least

after

U-chains.

study

method

We do not consider muscle

smooth

(see

synthesized

smooth

at the

in this

of the

morphological

of the collagen

supernatant

c311 lhydroxyproline

an absence

DISCUSSION: nature

BIOCHEMICAL AND BI~PHYSICAL RESEARCH COMMUNICATIONS

I and

III

to synthesize

652

(I,

II

and III).

a[ Aland

collagens interstitial

(24)

and

a[ B] -

whilst collagen

embryonic (35).

Vol. 84, No. 3, 1978

BIOCHEMICAL

AND BIdPHYSICAL

RESEARCH COMMUNICATIONS

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

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653