Skin cholesterol in ageing rats and experimental atheroma

PharmacologicaI Research Communications, Vol. 13, No. 3, 1981

241

SKIN CHOLESTEROl. IN AGEING RATS AND EXPERIMENTAL ATHEROMA. H. BOUISSOU +, J. DE CRAEVE ++, M. TH. PIERAGGI +, M. JULIAN +, J. C. THIERS +° +

Service d'Anatomie Pathologlque (Pr. H.Boulssou) C.H.U Rangueil Chemin du Vallon - 3IO54 Toulouse Cedex. ++ Service de Biochlmle (Pr. P.Valdigui~) C.H.U Rangueil - ChemJn du Vallon 31054 Toulouse Cedex. o Unit~ IO11NSERM - Biochlmie des L~pides (Pr.L.Douste-Blazy) Toulouse.

SUMMARY : The present experiments of skin cholesterol

have shown the following

in the rat, llke man, are related to age of the animal

and to hyperllpoproteinaemia.

2) In experimental

and a high fat diet) no relationship and the levels of skin cholesterol, KEY-WORDS

: I) The levels

atheroma

(BAPN : Ig/Kg/day

exists between the degree of atheroma in contrast

to the situation in man.

: Rat - Skin cholesterol - Ageing - Atheroma.

During normal ageing or arteriosclerosis,

the connective

aorta, coronary artery and human dermis are degraded (Bouissou, cholesterol

]980)

in a parallel way

(Bouissou et al, 1969, 1970) (Julian et al,

level has also been shown to be increased,

mis and the papillary dermis, and of aortic atheroma

1970). The skin

both in tile epider-

in relation to the degree of arteriosclerosis

(Bouissou et al, ]972,

al, 1976). Hyperlipoproteinaemia level (Ainati,

tissue of the

]973,

i974)

(Douste-Blazy et

produces a similar rise in skin cholesterol

1976).

Beta-aminopropionltrile

fumarate

(BAPN) administered

to 3-week-old white

Wistar rats for 9 weeks has been shown to cause dermal and aortic lesions similar to those seen in human ageing et al,

(Julian et al, 1973, 197]) (P~eraggi

I974). Although the animals are experimentally atheroresistant,

athe-

roma can be induced after BAPN treatment

(9 weeks) by feeding a high fat

diet for 42 weeks

1978) (Julian et al,

(Bouissou et al,

]978,

In order to correlate these observations, cholesterol

we have examined

]972,

1976).

the skin

levels of the rat during ageing and in experimental

amheroma to

determine if they are similar to the changes seen in the human. Our experiments

show that skin cholesterol

in the rat increases with age

and in rat fed a high fat diet, but is not changed

0031-6989/81/030241--09/$02.00/0

in experimental atheroma.

© 1981 The Italian Pharmacoloaical Society

PharmacoloEical Research Communications, Vol. 13, No. & 1981

242

MATERIAL AND METHODS and divided

:

White Wistar rats

into two groups

(3 weeks) were used in this study

:

Group I : To study skin cholesterol subdivided

into four

C

-" Control.

B

-

to diet were

:

Rats given BAPN.

D

- Rats fed a high fat diet.

B+I) - Rats g i v e n BAPN and h i g h Croup 2 : S k i n c h o l e s t e r o l increase with

:]Re,

in order

were used and k i l l e d

high fat

diet

complex

to study

fats

i n t h e (;roul~ I c o n t r o l

this at

4.5%,

In other cases

3, 5 and 7 months

magnetic

wool and rinsed

obtained

I0 m o n t h s .

(Rose et al,

The

~ l l u c o s e IO.3%, s t a r c h component

13.4%,

IO%, v i t a m i n

for t h e

of the animals

rats

in Group

had to he sacrificed

excess

and stored at -20 ° for choles-

liquid

as above.

(Liebermann, Aortic

through glass

for

to dry-

I hour in

The dry lipid extract

and cholesterol

1885). Cholesterol

lipid was extracted

(about

I hour on a

and evaporated

The sample was again stirred

in 2.5 ml chloroform,

fresh tissue.

weighed

(7/II v/v) for

15 ml chloroform

rinsed and ewllmrated

the method of Liebermann

removed,

1965). The sample was filtered

into a flask with

filtered,

tube,

treatment.

was redissolved

as mmol/IOOg

7, 8 and

[ o r 7 months e x c e p t

taken from each animal

ness on a rotary evaporator. chloroform,

70 r a t s

i n more d e t a i l .

1.8%).

in 15 ml chloroform/methanol

stirrer

were shown to

five auimal,~ from each suh-group were killed

Samples were thawed,

40 rag) and placed

6,

5%, m i n e r a l

sodi.m cholate

was a d m i n i s t e r e d

Skin samples were terol assay.

5.

( C a s e i n 202,

l-B,wh[cll hecause of the poor cmldition at 5 months.

increase

3, 4,

35%, c e l l u l o s e

IZ, c h o l e s t e r o l

The t r e a t m e n t

diet.

IR/KR/day f t l r 5 d a y s e a c h week by g a s t r i c

was t h e U.A.R. d i e t

a n i m a l and v e g e t a l

fat

levels

(I0 at a time)

BAPN was ~ i v e n a t

after

levels in response

level

assayed

by

is expressed

and determined

in the

same way.

RESULTS : Group I. Skin cholesterol the control

animals

7 months t h a n a t

cholesterol

levels

on plolon~ed

levels compared

o f tile f o u r s u b - g r o u p s a r e shown in T a b l e I .

In

(C) i t

can be s e e n t h a t

at

3, b u t

in skin cholesterol creased

levels

that

administration

skin cholesterol of

BAPN had no e f f e c t

(B). Rats fed on a high fat diet which

B + I) had significantly

to C and B, but was similar

to D.

on s k i n

(D) showed an increase

reached a maximum at 3 months,

treatment.

is highest

but was not inblgher cholesterol

Pharmacological Research Cornrnunications, VoL 13, No. 3, 1981

Fig. I - Normal aorta. The endothelial cells abut against the inner elastic laminae. The parallel elastic lamellae of the media enclose regular spaces containing muscle cells. Stain Masson ( X 25).

Fig. 2 - Five months BAPN intoxication. Typical vascular lesions of chronic lathyrism. In the inner half of the media, the elastic lamellae are thin, disrupted. stain Verhoeff ( X 40).

243

Pharmacological Research Communications, VoL 13, No. 3, 1981

244 l'All,I

I .

L'~AMIIP

Or II|ll

C M O L l l l r l ~ l i O l , I T I I I U D E I I ' ~ * I T 111J~ m l ' J r l l l l ~

C 0.8% ~ 0 , ~ 7 0 . ~ o l ~ o.oal • ,~ ~ o,o~

0 o o

0 . 9 ~ ! O,O7!

0,gP'

1.153 ~ 0.6~] t , ~ s l : o,o~o

a,l~ ~ sJI I

! 0.g67

9.~ d

I,NI

1,1.~ : O.~ll

o • p < o,o} l!

g

P

0

0 o

S.%'

*,*l ~

b .

I'D

p ( O.Ol

~,|9 b

0

0.*1 l *

o

c * p ( o,HI

• wa i l l * l l l l l | l .

~*o!

~ |¢ |,lord

d • p •

0,O~l

l.,|ll|wI*

t*.,l, .I .il. tk.tI~t,rel *t ~ 10.,hi ll,~l, *11,~ .~i**i,), I.Ik*fJ~.

No differences control

rats

Ii(~

v*~4a

{* IlolllOOl*

were seen in c])plesterol

a t 3,

5 and 7 months

levels in tlae aorta's qf the

(Table II),

and,histologlcal

of tile ac>rta wall showed no s.iRns (~Ii stt~uctura| modification BAI'N treatment rol

levels

Parietal

in the aorta at 3 months,

lesions,

logical fat

(g) showed a signil'ieant

samples

diet

characteristic from a l l

(p< 0.0OI)

and a t

time there greater (Fig.

or 3).

;nere:lse

5 and 7 m o n t h s

I n tl + I1, t h e o v e r C and 1; a t

treated

]evet

of ~ o r t i c

level

Atherosclerosis

cellular the

(three

or

four

same t h a n t i m s t a g e

llpids II

( . r o u p 2. The l e v e l s Table

III.

With age

wall

lesion

there

a~ 3 m o n t h s

accumulation

to D. At t h i s

in D at'3

with

was o b s e r v e d

spaces)

increased

overload

cholesterol

showed i n t h e

interlamellar

(,r e x i t , c e l l u l a r

levels

lipidic

found

of aortic

B. C and I}. T h i s c o i n c i d e s

the entire

in histo-

Rats fed a high

when c o m p a r e d

t o tile s i t u a t i o n

tile

(Fig. aorta

2).

was s i g n i f i c a n t l y

similar

an a t h e r o m a

layer

cholesterol

lesser

over

dia

in aorta

ch,,lesterol

increased

thoracic

were s e e n

BAPN ( F i g .

a t h e r o m a o n l y a~l e n d o t h e l i a l

4).

in choleste-

change at 5 months.

with

i s no a o r t i c area,

1).

( p < O.OOOI).

gnlficantly

the

lathyrism,

3 m o n t h s , b u t was n~ s i g n i f i c a n t

At 5 a n d 7 m m t t h s ,

(Fig.

increase

but no significant

of chronic

animals

(19) showed a l a r g e

(p
examination

months

in B + D is

in all

lesions

and f i b r o s i s .

si-

the appearance animals.

i n t i m a and i n the

important

of

inner

with

This

of In me-

intra-

lesion

was

o f human a t h e r o m a . of skin

choIesterol

is a significant

of each rat

(p/O.O01)

are

increase

shown i n in skin cho-

PharmacologicaI Research Communications, VoL 13. No. 3, 1981

2 45

, + i L l I i - c o * e , l l * O l a t l O l l I ( elOL+It|IgL I I I t U l l l l l l ! l l l t 1111111 ¢OlllOLl. f i l l , D i l l A l l I I + I I I I I + 11 - S - I M I I b I + +

C 0 . ~ 0 .+ 0.0~3 o . 1 ~ .+ O.oJ+ o,llo + o.oli

0.~9

~

a+i3

O

0 o o

! 0.0~1

+.~'

0

0 . 8 9 5 ~ ~.1~3

S.~'

0

o.?11 ~ O,G41 o.I¢1 ~ O.+l~

If,to 4 ll.|o 4

0 O

0.960 ~ 0.015

14.~ ~

12.~1~

0.~6""

0

B + O I,Sll J O.l,S P,?ol ~ O , | l i

I f . IS l II.11 d.

Jl.l li

l,l+ i ).ll d

0 0

•*

• P I o.a~ *, Jll,;rl,,i.

• • * ¢ o.ool ,,+*

v - I < q+oool : II,,lifd l,,laclei.

Levels *( el*t|¢ ckelucer*l 81 ) l e | t ~ m |JmlIS , I J e 4 i v I b , r J ) i +~ I . + . l h . tlt|+t++JJ.r~ .J0.~ *.oh+e,) +.d 61P m,stJ+ t+ial| +l~.l

lesterol, lute

confirming

levels

Group

the

of cholesterol

I controls

and this

results

found

in control

at

5 and

7 months are

3,

may b e d u e t o t h e

two different groups Studied at different

face

rats

: In man there is a n

status of hyperlipoproteinemia

increase

alterations

to the cells

lower than

rats

belonged

to

in both groups.

in skin cholesterol

(Ain't], 1 9 7 6 ) ,

1980). These variations

I . The a b s o -

times, In spite of this fact the

rosis (normal or rapid ageing of the, arteries) (Bouissou,

slightly

that the

increase in skin cholesterol • with ag e was paral]el DISCUSSION

in Group

in idiopathic

by the degree of arterioscleand atl~erosclerosis

in skin cholesterol are accompanied by

and tile intercellular matrix of dermal connective

tissues. The f[broblasts become qui~,scent, the elastic f~bres are few in number,

the collagen bundles are thin and the ground substance

(Bouissou et al,

1980,

These biochemical

1973,

(Ghomeln et al,

facts are probably due to the

1979)

1973) which is responsible

of the connective

(Narayanan et al,

1973)(Stein et al,

for tile formation and maintenance

ctal,

1978)(Goldstein

found

way w i t h

i n man.

are

However,

et al,

1977)(Orci et

1970).

In the rat we have shown that skin cholesterol significant

1978)

tissue of tlte dermis and for tile metabolism of choleste-

rol (Base eL ,al, 1977)(Brunzell al,

1976).

and histopatholt~gfcal

state of the fibroblast (Robert et al,

1974,

is abundant

and with

,a h i g h

i n man t h e r e

exists

fat

diet

increases

in a highly

in a similar

a relationship

way t o t h a t

between

the

in-

246

Pharmacologica/ Research Communications, Volo 13, No. 3, 1981

Fig. 3 - Three months high fat diet alone. Simple endothelial lipidic overload. Stain Masson ( X 40).

Fig. 4 - Five months of BAPN and high fat diet. Aortic atherosclerosis lesions. Destruction of the innermost elastic laminae, fibrosis, accumulation of foam cells and deposits of interstitial llplds in the media. Stain Masson ( X 25).

247

Pharmacological Research Communications, VoL 13, No. 3, 1981 Tlbl~ 111

Heart 0¢ skin ¢holeltero| |lrvl| f = l t o v | n l the ale of the : l t J ,

I

Ac[~ (,m,che)

~

~

~

6

B

I0

0,874 s

0.929

0.890

0.96?

1.0~0

1.0)2

1ol66

0,846

0.792

0,944

0,977

0.918

0.980

I.O0]

O.BO0

0.890

0.921

0,;66

0.841

0.690

0.690

0.769

O.A$1

0.79~

0.929

0.87~

0.13~

0,8;~

0,1~6

0,)02

0,8&$

O.BTT

0.823

1.011

0.679

0.810

0.7S3

0,806

0.119

0.820

0.9~

1,1&a

0.906

O.la5

0,?9l

0.704

0.812

0.9)9

0.94~ O,BSl

0.?)4

O.)J~

0.t74

0.117

0.85&

0.989

O.l?O

0.B¢6

0,699

0.921

0,903

0.95~

I.Oll

O.?l&

0.810

O.;9Z

0.791

0.8)1

0,86~

0,911

•

0.801

0.82)

0,8)2

0.64S

0.880

0,9)~

0.97Z

|

0.0S I

0,0; I

0.071

0.09~

O.OfiS

0.082

O. I0)

k i t e in ~ I I I O 0 1 ; f r * s h t i l I u e ,

•

~

• - man

I

-

stm~dlrd d f v i a t | o n

~:ch number in ~ach ¢oltstn rsptesentl one an|IS|. Ale correlation

-

| k i n cholesterol :

r - 0,$8), I - S,92"* ( 0 . 0 0 !

crease in dermal cholesterol and the intensity of the atheroma of

the p r e s e n t

experiments

show t h a t

it

is nat

t h e same i n t h e r a t .

Comparison of B + D with t) (Table I) shows that a t i s no s i g n i f i c a n t histological

increase

sections

is significantly

The d i f f e r e n c e different and t h e

aetiqn

rats

it

Firstly,

and t h e l e v e l

('Fable I I ) .

is significantly

in c o n t r a s t ,

o f BAPN on t h e

fibroblasts

in skin

a r e ; l l w a y s t h e same.

to u n d e r s t a n i l

is necessary BAI'N a c t s

mals,

tissues

!ipids

the differing

to consider

(Bouissou et al,

has membrane r e c e p t o r s

late

(Julian

at

action

these

3 months,

the

level

tissue

which allows

o f young r a t s

et al,

1979).

1979).

Secondly,

them t o a c c e p t

i t on t o t h e H.D.I. ( C o l d s t e i n

since

the cellular

of the fibroblasts

1978)(Jullan et al,

times,

by a

the diet

o f BAPN on young: and m a t u r e

tim r o l e o f BAPN a t

on d e r m a l c o n n e c t i v e

BAPN has no a c t i o n

L.D.L ahd p a s s

at

cholesterol

r~,i.~ed in B + D when compared t o D.

p e r i o d o f g r o w t h and c a u s e s m o d i f i c a t i o n s stitial

of aortic

even t h o u g h

b e t w e e n 3 months and 5 o r 7 m o n t h s may be e x p l a i n e d

increase

In o r d e r

5 and 7 months there

tile l e v e l o f s k i n c h o l e s t e r o l

show a t h e r o m a ,

increased

of skin cholesterol

i.

; the results

et al,

that BAPN also would act on these receptors

level.

during

the

and t h e

inter-

In m a t u r e a n i -

the fibroblast

the cholesterol

of

the

1977). One may p o s t u -

in a transitory manner,

only during the period of growth. At 3 months,

the level of skin cholesterol

in B + D was significantly

different to tbose rats given only a high fat diet. A t this stage in the development of the rat, BAPN would affect the cholesterol membrane recep-

248

Pharmacological Research Communications, VoL 13, No. 3, 1981

tors

~f tile f i b r o b l a s t s

lead

to an i n c r a e s e d

high fat diet

and the

skin cholestertd.

during the

cholesterol

over

cholesterol

fmmd

The i n c r e a s e

anlmals,

BAPN. The i n c r e a s e d skin cholesterol,

lipid

lipid

but

an e f f e c t

to the uxtrm:ellular The a c t i o l l o f

hut

in t h e s k i n o f a n i m a l s

in s k i n c h o l e s t e r o l

it

is only

on t h e matrix

fected

by BAPN, and d u r i n g

conteut

=~f t h e d i e t . l e a d s

and human t i s s u e s

rimental level

atheroma,

the

of t h e r e c e p t o r s the rat

increased

skin cholesterol.

is af-

lipid

At l a t e r

stages

of t h e c e l l

t o BAPN i s

c o u l d be a t t r l b u t e d

to ageing

the high fat

stages

in m a t u r e r a t s

T h i s ellallgt, ill a o r t i c

diet

leads

o f d e v e l o p m e n t b u t BAPN

as a consequence of cholesterol

the differing

levels

reactions

the apwas n o t

of the rat

t o t h e a p p e a r a n c e o f an a t h e r o m a . have shown t h a t

between skln cholesterol, however°

of skin cholesterol

parallel

stage

In t h e c a s t ' o f t h e a o r t a

CONCLUSION : T h e s e e x p e r i m e n t s is a relationship

in t h e membrane i s n o t

the sensitivity

in t h e d e r m i s and d e m o n s t r a t e s

in relat-ion

metabolism.

t h e d e v e l o p m e n t of

at all

that

function

the capacity

developmental

eholesterul

(3 m o n t h s )

in c h o l e s t e r o l

in

in s k i n c h o l e s t e r o l

level

in g + D and D i s a

its

receptors

to an e l e v a t e d

the cholesterol

p e a r a n c e t~f an a t h e r o m a . mirrored

role

alterntion

in t h e d e v e l c , p m e n t (5 and 7 m o n t h s )

increased

of

by the p r e s e n c e o f

affecting

and also, i t s

L.I).i. or /t.D.I, o r b o t h d u r i n g

in aortic

levels

BAPN

for

t o an i n c r e a s e

of the

iU t h e young a n i m a l s

either

diet.

no e l e v a t l o n

in skin

of

i t would a p p e a r t h a t

and t h e h i g h l i p i d

to an i n c r e a s e

and

in B + D and D l'eads to a n e l e v a t i o n

BAPN on t h e c h o l e s t e r o l

increase

leads

is unaffected

known, but

r e d u c e d and t h e

would

fed a h i g h f a t d i e t .

fibrM~last,

this

i.n t h e d i e t

of tile l a t h y r o g e n

['n m a t u r e a n i m a l s

diet- and

intake

intake

Continuation

d e v e l o p m e n t of r a t s

tile c o n t r o l

consequence of the high

would e x e r t

incre¢lsed lipid

relationship

showed t h a t

in the rat,

a g e and h y p e r l i p l d a e m l a .

no r e l a t i o n s h i p

and tl~e d e g r e e o f a t h e r o m a ,

exists

l i k e man,

between skin cholesterol

exists

there Expe-

between the

u n l i k e man where a levels

and t h e d e g r e e

o f atheroma.

REFFRENCES : AINATI M, l"h~.se M0decine Toulouse, 1976, N ° 472. BASU S. K, ANDERSON R.G.W, GOI.DSTEIN .I.L, BROWN H.S, J. Cell Biol, 74, 119-135. BOUISSOU H, Medlcographia, 1980, 2, 9-15. BOUISSOU II, FABRE H. TII, JULIAN M, RUMEAU 3.L, Pres. Ned, 1969, 77, BOUISSOU II, FABRE H. TII, JULIAN H, RUMEAU 3.L, HURON R, Rev. Europ. Clin. Biol, 1970, 15, 444-453. BOUISSOU 11, PIERAGGI M. Tll, JULIAN H, PENDARIES I. SORBARA R, Folla logica,

1972, 20, 2 7 1 - 2 7 8 .

1977,

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