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Det här verket är upphovrättskyddat enligt Lagen (1960:729) om upphovsrätt till litterära och

konstnärliga verk. Det har digitaliserats med stöd av Kap. 1, 16 § första stycket p 1, för

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/éO

Endothelial Integrity and Cholesterol

Transfer in the Aorta of the Rabbit

Cholesterol content, accumulation and elimination in morphologically

defined experimental atherosclerotic lesions and normal tissue

By

G Ö R A N B O N D J E R S

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Endothelial Integrity and Cholesterol

Transfer in the Aorta of the Rabbit

Cholesterol content, accumulation and elimination in mo rphologically

defined experimental atherosclerotic lesions and normal tissue

AKADEMISK AVHANDLING

SOM FÖR VINNANDE AV MEDICINE DOKTORSGRAD, MED VEDERBÖRLIGT TILLSTÅND AV MEDICINSKA FAKULTETEN VID UNIVERSITETET I GÖTEBORG, KOMMER ATT OFFENTLIGEN

FÖRSVARAS Å AULAN, SAHLGRENSKA SJUKHUSET, ONSDAGEN DEN 24 MAJ 1972 KL. 9 F.M.

A v

G Ö R A N B O N D J E R S

MED. KAND.

GÖTEBORG 1972

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University of Göteborg, Göteborg, Sweden.

Endothelial Integrity and Cholesterol

Transfer in the Aorta of the Rabbit

Cholesterol content, accumulation and elimination in morphologically

defined experimental atherosclerotic lesions and normal tissue

By

G Ö R A N B O N D J E R S

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2

The present thesis is based upo n the following research papers:

I . BONDJERS, G., and S. BJÜRKERUD: Fluorometric determination of cholesterol and cho lesteryl ester in tissue on the nanogram lev el. Anal. Biochem. 42:

363 (1971 ).

II. BJÜRKERUD, S., and G. BONDJERS: Endothelial integrity and viability in the aorta of the normal rabbit and r at as evaluated with dye exclusio n tests and in terference contrast microscopy. Atherosclerosis J_5: 285 (1972). III. BONDJERS, G ., and S. BJÜRKERUD: Cholesterol accumulation and conte nt in

regions with defined endothelial integrity in the normal rabbit aorta. (1972). Accepted for publication in Atherosclerosis.

IV. BJÜRKERUD, S., and G. BONDJERS: Arterial repair and at herosclerosis after mechanical injury. Part 1. Permeability and l ight microscopic characteris­ tics of endothelium in non-atherosclerotic and a therosclerotic lesions. Atherosclerosis _1_3: 355 (1971 ).

V. BONDJERS, G., and S. BJÜRKERUD: Arterial repair and at herosclerosis after mechanical injury. Part 3. Cholesterol accumulation and remov al in morpho­ logically defined regions of aortic atherosclerotic lesions in the rabbit. (1972). Accepted for publication in Atherosclerosis.

VI. BONDJERS, G., and S. BJÜRKERUD: Arterial repair and at herosclerosis after mechanical injury. Part 4. Uptake and composition o f cholesteryl ester in morphologically defined regions of atherosclerotic lesions. Atherosclerosis J_5: 273 (1972).

These papers w ill be re ferred to in the text by their roman numerals. Abbreviations: CEFA

LCAT TLC

-cholesteryl ester fatty acid

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1 INTRODUCTION 5

1.1 Atherogenesis 5

1.2 Déposition o f cho lesterol 5 1.3 The endothelium and atherogenesis 6 1.4 Experimental atherosc lerosis induced by mechani cal trauma 6 1.5 Purpose o f the study 7 2 SUMMARY O F T HE R ESULTS 8 2.1 Structura l and fun ctional aspects o f the normal a o r t i c

endothelium 8

2.2 Mechanisms f o r the transfer o f cho lesterol i n t o the normal

a o r t ic tissue 9

2.3 Structure o f the endothelium i n d i f f er e n t a or t i c lesions

induced by mechanic al i nj u r y i n the rabb it 9 2.4 Mechanisms f o r the accumulation o f chol esterol i n athero­

s cl e r ot i c lesions induced by mechan ical i n j ur y i n the rabb it 9 2.5 Cholesterol transfer i n morphologically defined regions of

a o r t i c experimental atheroscl erotic lesions induced by

superfi cial mechanical i n j ur y 10

3 METHODOLOGY 11

3.1 Experimental animals 11 3.2 Experimental atherosclero sis 11 3.3 Cholesterol determination and fra ctionat ion 12

3.4 Morphology 13

3.5 Studies with labelled cho lesterol 14 3.6 S t a t i s t i c a l methods 16 4 ENDOTHELIAL INTEGRITY 17 4.1 Endothelial i n t eg r it y i n the normal aorta 17 4.2 Permeability characte ristic s of normal a o r t ic endothelium 19 5 CHOLESTEROL TRANSFER 23 5.1 The o r i g in of cho lesterol i n the normal a rt e r i a l tissue 23 5.2 The o r i g in of excess cho lesterol i n athero sclero tic lesions 25 5.3 Cholestero l transfer i n a r t er i a l tissue with i n t a c t endo:

thelium 27

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PAGE

6

ENDOTHELIAL INTEGRITY AND EXPERIMENTAL ATHEROSCLEROSIS

37

6.1 Experimental atherosclerosis induced by mechanical trauma

37

6.2 Dietarily induced experimental atherosclerosis

37

6.3 Endothelial integrity and the filtration hypothesis

38

7

ABSTRACT

39

8

ACKNOWLEDGEMENTS

41

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s cl e r ot i c tissue (reviews: 2 , 25, 26, 27). These r e s u lt s have be en considered to be a strong argument i n favour o f the hypothesis that f i l t r a t i o n of plasma i s the primary facto r causing c holesterol deposition i n ath erosclerotic lesions ( 2, 25, 27).

1.3 The endothelium and atherogenesi s

The en dothelia l c e l l layer forms th e boundary betw een the blood and the suben­ do thelial a rt e r i a l tiss ue (review: 28). Thus, a l l substances which e nter the a r t er i a l tissue from th e lumen, must pass through the endothelium (28). By v i r t u e of t h e ir anatomical p os i t i on , the endotheli al c e l l s are also connected t o the formation o f mural thrombi (28, 29). I n addition , i t seems very l i k e l y tha t the endothelial c e l l layer should be the f i r s t struc ture influence d by increased hemodynamic f orce (29 ), f o r example due t o turbulance. I t follows that hypotheses on athero genesis, such as the f i l t r a t i o n hypothesis, the en­ crusta tion hypothesis, and the hemodynamic stress hypothesis, must take the propertie s of the endothelium i n t o account. However, the morphology o f the a r t e r i al endothelium i s d i f f i c u l t to study by me ans of hi stologica l sec tions, as i t i s arranged i n cylinde rs. Studies on en fac e preparations may, on t he other hand, pro vide more re presentative information on the structure o f the endo­ thelium i n normal and a therosclerotic a r t e r i es . The e laboration of new, simple techniques f o r the preparation o f such mat eria l (30), with decreased r i sk s f o r a r t i f a c t s , may, therefore , be o f great potentia l significance f o r the advance­ ment o f our knowledge o f atherosclerosis and i t s o ri g i n .

1.4 Experimental athe rosclerosis induced by mechan ical trauma

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w i th c h a r ac t e r i s t i c and rep roduci ble morphological propertie s have bee n i den­ t i f i e d by l i g h t microscopy (34) ,transm ission e l e ct r o n microscopy ( 3 5) , and scanning e l e c t r o n microscopy ( 3 6 ). These r e s u l ts i n d i c at e t h a t experimental a r t e r i a l lesions w i t h p r e di c t a b l e morphological p r o pe r t i e s and o f known age, may be pro duced a t w i l l by d efined mechanical i n j u r y .

The s e l e c t i o n o f m a t e r i a l may e x e r t a determining i n f l ue n c e on the relevance o f r e s u l t s obtained from morpholo gically heterogeneous o r undefined t i s s ue s . Variable composition o f the t i s su e samples inve stigated may obs cure t r u e charac­ t e r i s t i c s ( 2 7 , 3 7 ) , and a t the same time produce a r t i f a c t u a l r e s u l t s ( 3 7 ) . These con siderations may be e s p e c i a l l y relev ant f or studies on a t h e r o s cl e r ot i c lesions which are f o c a l l y d i s t r i b u t e d , s t r u c t u r a l l y v a r i a b l e , o f d i f f e r i n g age, and have a mar ked i n t r i n s i c heterogeneity (reviews: 2 , 3 ) . Therefore, the p o s s i b i l i t y o f inducing experimental a t h er o s c l e r o t i c lesions w i t h defined morphological p r o pe r t i e s by me ans o f mechanical i n j u r y may allow workers i n

t h i s f i e l d t o o b t ai n p a r t i c u l a r l y r e l e v a n t infor mation about biochemical and other p r o pe r t i e s o f a t h e r o s c l e r o s i s .

1 . 5 Purpose o f the study

The general aim o f the present i n ve s t i g a t i o n was t o study two f a c t o r s i n ti m a t e l y r e l a t e d t o curre nt concepts on atherogenes!'s, as discussed above: ( i ) s t ru c t u ra l and f u n c t i o n a l p r o p e r t i es o f the endothelium i n normal and a t h e ro s c l e r o t i c t i s s u e ; and ( i i ) l o c a l t r a n s f e r mechanisms f o r f r e e and e s t e r i f i e d chole sterol i n normal a r t e r ie s and i n a t h er o s cl e r o s i s . For t h i s purp ose i t was necessary t o develop new techniques f o r the study o f endothelial c e l l v i a b i l i t y ( I I ) , and mo re sen­ s i t i v e methods f o r the determina tion o f chole sterol i n t i s s ue ( I ) . These t ech­ niques were com bined and applied t o normal aorta and morpho logically defined a t h e r os c l er o t i c lesions induced by mechan ical trauma i n normo-1ipidemi c r a b b i t s , i n an attempt t o c o nt r i b u t e t o the knowledge concerning the f o l l o w i n g more spe­ c i f i c problems:

1) I s the normal a o r t i c endothelium o f r a b b i t and r a t s t r uc t u r a l l y and func­ t i o n al l y homogeneous? ( I I , I I I ) .

2) What i s the nature o f the l o c a l mechanisms involve d i n the t r a n s f e r o f c h ol ­ e s t er o l i nt o the normal r a b b i t aorta? ( I I I ) .

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4) What local mechanisms ma y be involved i n the accumulation o f cho lesterol i n experimental atherosclerotic lesions induced by defined mechanical i n j u r y i n the rabbit ? (V).

5) Are th e chara cteristics o f accumulation, removal and composition o f free and e s t e r i f i ed cholesterol rel ated t o selecte d morphological properties o f mechanically induced at herosclerotic lesions i n the rabbit ? (V, V I ) .

2 , S U M M A R Y O F T H E R E S U L T S

2.1 Structura l and fun ctional aspects o f the normal a o r t ic endothelium 2.1.1 Structure o f the normal a o r t i c endothelium

11 : Cell v i a b i l i t y i n ra bbit and r a t aorta was evaluated by me ans o f dye exc lu­ sion t e s t s , which re sulted i n a d i f f er e n t i a l sta ining o f the a o r t ic surface. Unstained areas were covered w ith normal, i nt a c t endothelial c e l l s. Stained areas were covered w ith defective endothelium, containing endothelial c e l l s with struc tural properties, ind icatin g i r r e v e rs i b le i n j u r y or c e l l death. Micro-thrombi were frequen tly seen adhering t o inj ured endothelial c e l l s. Medial smooth muscle c el l s underlying defective endothelium wer e a lso i nj u r e d , as judged from t h e ir morphological and s taining characte ristic s. The presence o f injured endo­ thelium and medi a c e l l s i n regions, where increased hemodynamic s t r a i n i s l i k e ­ l y to occur, and which constitute predilectio n sit es f o r atheroscleros is, i n di ­ cate that locali zed areas i n the normal art ery may be precond itioned f or add i­ t i on a l i n j u r y leadin g t o atherosclero sis.

2.1.2 Permeability o f the normal a o r t ic endothelium

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10 2 . 5 C h o l e s t e r o l t r a n s f e r i n m o r p h o l o g i c a l l y d e f i n e d r e g i o n s o f a o r t i c e x p e r i ­ m e n t a l a t h e r o s c l e r o t i c l e s i o n s i n d u c e d b y s u p e r f i c i a l m e c h a n i c a l i n j u r y 2 . 5 . 1 C h o l e s t e r o l t r a n s f e r a n d c h o l e s t e r o l c o n t e n t _V: D i f f e r e n t r e g i o n s h a v i n g c h a r a c t e r i s t i c a n d r e p r o d u c i b l e m o r p h o l o g i c a l p r o ­ p e r t i e s m a y b e r e c o g n i z e d i n a t h e r o s c l e r o t i c l e s i o n s i n d u c e d b y s u p e r f i c i a l m e c h a n i c a l i n j u r y w i t h l a r g e a r e a . T h e c h o l e s t e r o l c o n t e n t i n r e g i o n s w i t h d e ­ f e c t i v e e n d o t h e l i u m w a s 3 t i m e s t h a t o f t h e c o n t r o l s , w h i l e , o n t h e o t h e r h a n d , t h e c h o l e s t e r o l c o n t e n t i n r e g i o n s , w h e r e t h e o r i g i n a l a r t e r i a l w a l l s t r u c t u r e h a d b e e n r e s t o r e d , w a s e q u a l t o t h a t o f t h e c o n t r o l s . T h i s i n d i c a t e s t h a t c h o l ­ e s t e r o l i s e l i m i n a t e d d u r i n g r e p a i r a n d r e - e n d o t h e l i a l i z a t i o n o f t h e l e s i o n s . I n r e g i o n s w i t h d e f e c t i v e e n d o t h e l i u m , t h e s p e c i f i c a c t i v i t y o f c h o l e s t e r y l e s t e r w a s l o w e r t h a n t h a t o f f r e e c h o l e s t e r o l , s u g g e s t i n g t h a t f a r m o r e c h o l ­ e s t e r o l e n t e r s t h e a r t e r i a l w a l l t h a n i s d e p o s i t e d i n i t . T h e s e r e s u l t s f u r t h e r s u p p o r t s u g g e s t i o n s t h a t c h o l e s t e r o l m a y b e r a p i d l y e l i m i n a t e d f r o m t h e a o r t i c t i s s u e . 2 . 5 . 2 E s t e r i f i e d c h o l e s t e r o l

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3 . M E T H O D O L O G Y

3.1 Experimental animals 3 . 1. 1 Rabbits

I n a systematic study i n vo l v i n g a l a r g e number o f animals, i t was found t h a t a l b i n o r a bb i t s o f the Danish co untry s t r a i n have a very low incide nce o f a r t e r i o s c l e r o t i c changes i n the descending t h o ra c i c and abdo minal aorta ( 3 9 ) . Mal e r a bb i t s o f t h i s s t r a i n , obtained from on e bree der, fed 125 gms d a i l y o f l o w - c al o r i e , high f i b e r - c o n te n t r a b b i t p e l l e t s , and weighing 2 . 4 - 3 . 5 k g , were used i n I I , I I I , V an d VI. Serum chol esterol l e v e l s o f the animals used f o r chole sterol t r a ns f e r experiments were 27-55 mg/100 m l . I n I V, female, a l b i n o r a bb i t s o f the Swedish country s t r a i n , obtained from one breeder, fed ad 1 i b i -tum on ordin ary r a b b i t p e l l e t s , and weighing 3 . 6 -4 . 3 k g , were used. The r e s ul t s from IV have bee n confirmed by studies on male, a l b i n o r a bb i t s o f the Danish country s t r a i n , both w i t h l i g h t microscopy ( 3 4 ) , transmission e l e c t r o n micro ­ scopy ( 3 5 ) , and scanning e l e c t r o n microscopy ( 3 6 ) .

3 . 1 . 2 Rats

Male, Sprague-Dawley r a t s , fed ordinary r a t p el l e t s ad l i b i t u m , and weighing between 400 and 500 gms, were use d i n I and I I . They we re f r e e from chroni c r e s p i r a t o r y disease, as judged by inspection post mortem.

3.2 Experimental athero sclerosis

A t h er o s c l e r o t i c lesions were induced ( I V , V, VI) by me ans o f a s u p e r f i c i al mech­ anical i n j u r y w i t h l a r g e area (transverse i n j u r y ) w i t h the m icrosur gical i n s t r u ­ ment described by Björkerud (31). I n a study on the morphology o f t h i s type o f a t h e r o s c l er o t i c l e s i o n a t d i f f er e n t times a f t e r the operation, we found t h at a l a r g e segment o f the a o r t i c surface was i n i t i a l l y denuded o f endo thelial l i n i n g by t he trauma ( 3 4 ) . During r e - e n d o t h e l i al i z a t i o n o f t h i s d ef e c t , regions, w i t h d i f f e r e n t , s p e c i f i c morphological p r o p e r t i es developed i n the i n i t i a l l y i n j ur e d segment (34) (see schemat ic i l l u s t r a t i o n s ; V: F i g . 1 and V I : F i g . 1 ) . These regions c o n s t i t u t e consecutive stages i n the r e pa i r o f the t i s s u e ( 3 4 ) .

i - The c e n t ra l region o f the lesio ns i s moderately eleva ted, s t i l l devoid o f en­ d o t h e l i al l i n i n g , and conta ins abundant, f i n e l y dispersed, e xt r a c e l l u l a r l i p i d ( 3 4 ) . This region was designated YA RD ( 3 4 ) .

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the intima and t he media sh ow a varyin g degree o f n e cr o s i s , w i t h i n t r a c e l l u l a r l i p i d i n shoals o f foam c e l l s ( 3 4 ) . The mor phology o f such p arts i s very s i m i ­ l a r t o t h a t o f f a t t y streaks ( 3 4 ) . The whole regions were designated BANKS ( 3 4 ). i i i . I n some por tions o f the i n i t i a l l y i n j ur e d segment, the normal a r t e r i a l wall

s t r u c t ur e has been m ore o r less completely restored. The en dothelial l i n i n g i s continuous, and the i n t i m a i thicke ning has regressed i n such re gions ( 3 4 ) . They we re designated ST AGES ( 3 4 ) .

I n the cholestero l t r a n s fe r studies (V, V I ) defined samples o f YARD, non-end o-t h e l i a l i z e d BANK, e n d o o-t h e l i a l i z e d BANK an d ST AGE we re c uo-t ouo-t w i o-t h m i c r o s c i ss o r s, freed from adv ential t i s su e w i th m i c r o d i s s e ct i o n , and analyzed as described below.

3.3 Cholesterol determination and f r a c t i o na t i o n 3 . 3 .1 Cholesterol determina tion

Previous me thods f o r the determination o f chol esterol were e i t h e r not s e ns i t i v e enough ( f o r review: see e . g . 40) o r too laborious (discussion : see I ) , t a k i n g account f o r the l a r g e number o f very small t i s s u e samples. Therefore, a simple ultramicro-method f o r the determination o f f r e e and e s te r i f ie d cholestero l i n the nanogram ran ge wa s developed. I t i s described i n d e t a i l i n I . Optimal con­ d i t i o n s f o r the r e a c t i o n and pos sible i n t er f e r e n c e by a number o f other s t e r o l s were i n v e s ti g a t e d . The recovery o f chole sterol added t o an a r t e r i a l t i s s u e l i p i d e x t r a c t was 101.5±2.1 % (S.D.) ( I ) .

3 . 3 . 2 F r a c t i o n a t i o n o f f r e e and e s t e r i f i e d chole sterol

Free and e s t e r i f i e d chole sterol were separated ( I V , V, VI) by t h i n l a y e r chrom­ atography on a 250u l a y e r o f s i l i c a gel H, as described by Glo ster and Fle tcher ( 4 1 ) , w i th the s l i g h t l y modified s o l v e n t : n-hexane: d i e t h y l e t h e r ; a c e t i c a c i d ( 8 0 : 2 0 : 2 ) ( 4 2 ). The recovery o f added f r e e cholestero l was 99.0±1.9

I

( S . D . ) , and the recovery o f added c holesteryl e s t e r was 100.0±2 .7 % (S.D.) a f t e r TLC ( I ) . 3 . 3 .3 F r a c t i o n a t i o n o f d i f f e r e n t c h o le s t er y l ester classes

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f a t t y a c id s ; and f r a c t i o n 5 consisted mainly (ca. 90 % ) o f c h o le s t er y l ester s w i th penta- and hexa-unsaturated f a t t y acids . The recovery o f t o t a l choleste­ r y l e s t e r a f t e r t h i s f r a c t i o n a t i o n procedure wa s i n our hands 93.2±5.2 Ï (S.D.)

( V I ) , as determined by compar ison w i t h the t o t a l c h o le s t e r y l e s t e r f r a c t i o n obtained a f t e r TLC according t o Gloster and Fle tcher ( 4 1) , modified as described above.

3 . 3 .4 Recovery o f chol esterol

I n the assay o f m a te r i a l conta ining r a d i o a ct i v e c h o l e st e r o l , the r a d i o a c t i v i t y i n an a l i q uo t o f the o r i g i n a l washed t i s s u e l i p i d e x tr a c t was determin ed. The recovery o f t o t a l r a d i o a c t i v e chole sterol a f t e r f r a c t io n a t i o n , e x tr a c t i o n from the s i l i c a g e l , and washing, was 98.0±4.2 / (S.D.) ( I I ) and 96.1±6.8 % (S.D.) ( V ) . 3.4 Morphology

3 . 4 . 1 C e l l v i a b i l i t y t e s t s

Cell v i a b i l i t y i n the a o r t i c endothelium w as evaluated ( I I , I I I , V, VI) by dye exclusion t e s t s as described i n I I . I n p r e li m i n a r y experiments we tested a num ­ ber o f dyes used by othe rs f or c e l l v i a b i l i t y t e s t s ( f o r referen ce: see 4 5 ) . The dyes were: lissamine green, erythrosin e A, erythrosin e B, water-s oluble e o s i n , trypan blue and n i g r o s i n . Only n i g r o si n and try pan blue y i e l d e d s a t i s ­ f a c t o r y contr ast and s t a i n i n g i n t e n si t y . The t i s s u e d i s t r i b u t i o n o f these s t a i n s on the l i g h t microscopical l e v e l was studie d i n d e t a i l , as w e l l as t h a t o f the non-to xic s t r u ct u r a l isomer o f trypan b l u e , Evans b lue. The general s t a i n i n g p a t te r n o f these com pounds w as s i m i l a r , and those o f trypan blue and Eva ns blue were almost i d e n t i c a l . Uncomplexed Ev ans blue under these co nditions has e n t i r e l y d i f f er e n t s t a i n i n g p r o pe r t i e s compared t o Evans blue used i n vivo ( I V ) . Under i n vivo conditi ons Evans blue i s complexed t o albumin (46) and may be us ed as a marker f o r increased pe rmeability f o r albumin through the endothelium (review:46 ). 3 . 4 . 2 S i l v er s t a i n i ng and pe rmeability f o r plasma p rotein s

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I n view o f the r e s u l t s discussed abov e, i t was i n f e r r e d t h at observations a t more than one t ime would not c o nt r i b u t e s i g n i f i c a n t l y t o the inform ation o b t ai n ­ ed from o nly one time period a f t e r the i n j e c t i o n o f the isoto pe. I n order t o o b t a i n maximal r a d i o a c t i v i t y i n the t i ss u e , before e q ui l i b r i u m s pe c i f i c a c t i ­ v i t i e s between the t i s s u e cholestero l pools and the blood c holesterol p o ol , the animals were k i l l e d 24 hours a f t e r the i n j e c t i o n o f the isotope. However, due t o the d i f f i c u l t y i n d e fi n i n g i n p r e ci s e , absolute terms the c o nt r i b u t i on s o f actual t r a n s f e r and exch ange t o the amount o f l a b e l l ed c h o l e s t e r o l , we r e ­ f r a in e d from expressing the r a d i o a c t i v i t y measurements i n terms o f chole sterol amounts o r serum equiv alents. The r a d i o a c t i v i t y i n the t i s s u e samples a f t e r

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24 h r o f exposure t o l a b e l l ed cholestero l was designated UPTAKE o f H-choles t-e r o l , and wa s t-exprt-esst-e d as r a d i o a c t i v i t y pt-er u n i t surfact-e art-ea.

3 . 5 . 5 L i q ui d s c i n t i l l a t i o n counting

The r a d i o a c t i v i t y o f the samples wa s determined i n a l i qu o ts o f t o t a l , f r e e and e s t e r i f i e d chole sterol i n a Packa rd Tri- Carb L i q u i d S c i n t i l l a t i o n spectropho­ tometer, w i th a s c i n t i l l a t i o n mixture o f 0.5 % butyl-PBD ( w / v ; CIBA L t d . , Basle, Switzerland) i n toluene . The samples wer e counted f o r a t l e a s t ten minutes o r i n l o w - a c t i vi t y samples t o a t h e o r e t i c a l standard d e v i a ti o n o f less than 2.5 % (more than 2000 r e g i s t e r e d counts)(59). Quenching wa s corrected f o r by m eans o f an extern al standard.

3.6 S t a t i s t i c a l methods 3 . 6 . 1 D i s t r i b u t i o n o f the data

There were no a p r i o r i c r i t e r i a i n di c a t i n g t h a t the numerical data o f our s t u ­ dies were normally d i s t r i b ut e d . I n some inst ances, there was eve n s trong evidence i n d i c a t i n g t h a t there was a skewed d i s t r i b u t i o n (see f o r example I I I : F i g. 2 ). I n view o f the questionable v a l i d i t y o f the s i g n i f i c an c e l i m i t s o f the student's t e s t and oth er t e s t s f o r normally d i s t r i b u t e d populations i n populations w i t h unknown d i s t r i b u t i o n ( 6 0 ) , d i s t r i b u t i o n - f r e e (non-parametric) t e s t s were used throughout t he study.

3 . 6 . 2 Significance t e s t s

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If more than one sample of either type was present, all possible differences were calculated and ranked for Wilcoxon's test. In V, tissue samples from the same lesions were paired.

3.6.3 Regression analysis

Correlations were tested with Spearman's rank correlation coefficient (62).

3.6.4 Considerations on the number of samples

A large number of samples was investigated in III and V, although the total number of animals was restricted. In addition, the samples were evenly distri­ buted between the animals, and no single animal deviated from the general trend of the observations. Therefore, the possible uncertainty introduced by the small number of animals does not decrease the validity of the conclusions in the studies.

4 , E N D O T H E L I A L I N T E G R I T Y

4.1 Endothelial integrity in the normal aorta

4.1.1 Cell viability tests

Although there is strong evidence for endothelial changes leading to increased endothelial permeability as one central factor in atherogenesis (28, 63), only little information has been available on the structural integrity of normal arterial endothelium. The results from the application of dye exclusion tests to test endothelial cell viability, indicated a marked structural heterogeneity in the normal aortic endothelium (II). Areas stained with nigrosin, trypan blue and Evans blue were present in the aortae of rabbits and rats, most fre­ quently at branching points and in the aortic arch, i.e. in regions where in­ creased hemodynamic strain may be expected.

4.1.2 Intact endothelium

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i n the rabb it aorta and other ar te ries o f simila r size (reference: 28 ) . Studies of the incorporation of lab elled thymidine have in dicated that the t ur n­ over o f endothelial c e l l s i n the normal aorta i s very low (67 ), and i t has even been suggest ed t ha t these c e ll s are replaced only a few times i n an anim al's life sp an (67).

4.1.3 Defective endothelium

The concept o f a continuous endothelial c el l l ay er , completely covering the lumenal surface, may ser iously be questioned against the back-ground o f the results of I I . The ex istence of regions with aberrant str uctur al or functional properties i s suggested also by t he recent reports o f iso lated areas w ith an increased rate o f c el l divisi ons i n the aorta o f guinea-pigs (68, 69, 70). Such areas were lo ca lized t o the a o r t ic arch and th e mouths o f a o r t ic branches

(68, 69, 70), and, a f t er local experimental i n j u r y , t o the damaged regions (70). The i n t r ac el l u l a r uptake o f anionic dyes, such as nigro sin, trypan blue, or Evans blue, i s probably related t o a decrease d i n t e g r i t y o f the c el l membrane during c e l l ul a r i nj u r y or death (45, 71). I n the interference contras t micro­ scope, stained c e l l s i n transparent samples o f normal r ab b i t aorta were ch arac­ ter ized by c ytoplasmic and nucl ear oedema, i n t r a ce l l u l a r vacuolization, and changed surface properties ( I I ) . I n a systematic study o f the morphology o f injured and dead c e l l s o f other types i n tissue c u l t u r e , Bessis found t ha t these chara cteristic s are reproducible components o f pre- and perim ortal changes i n a variety of d if f e re n t c e l l s (71). The in duction o f a local mechanical i n j u r y , or chemical i n j u r y , i s followed by similar alterat ions i n vivo i n endothelial c el l s , as i ndicated by l i g h t microscopy o f sectioned samples and tran smission electron microscopy (r eview and references : 63). Furthermore, swelling and c yto-l y s i s have been described as components o f spontaneous c eyto-l yto-l death i n a r t e ri a yto-l endothelium (72). A l l t h i s evidence in dicate s tha t increased uptake o f supra­ v i t a l stain i n sp ecific regions o f the a o r t i c endothelium i s due t o an in cre-sed incidence o f damaged or dead c e ll s i n these re gions.

4.1.4 Endothelial i n t e g r i t y and he modynamic s t r ai n

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a o r t ic wall was r e s tr i c t ed under these conditions. On the other hand, s t r e t ­ ching o f the aorta considerably increased the passage of lab elled albumin i n t o the tissue (86), indicating derangement o f a b a r r i e r against f i l t r a t i o n o f plas­ ma c onstitu ent s. There i s considerable evidence that stretching o f the aorta may decr ease end othelial i n t eg r it y (e.g. 87 ), and ther efore these results are well compatible with the idea that the maintena nce o f endo thelial conti nuity may prevent excessive i n f l u x o f plasma c onstituents i n t o the a o r t i c tis sue. 4.2.3 Increased e ndothelial perm eability

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As sugge sted by Duncan' s study on th e passage of I-albumin i nt o the normal a o r t i c tissue of the dog (87, see above), the formation o f discontinuitie s i n the a o r t i c endothelium m ay lead t o increased p ermeability f o r plasma p roteins. I n accordance w ith such a me chanism increased p erm eability to the Evans blue albumin com plex was confine d t o regions o f a or t i c lesions devoid o f endothel­ i a l l i n i n g , a ft e r the induction o f a d efined mechanical i n j u r y t o the aorta o f normal rab bits ( I V) . Not o nly local mechanical i n j u r y but also generally increa­ sed hemodynamic stress due to the development o f hypertension, may induce d i s ­ cont inuity i n the a r t e r i a l endothelium (88, 89, 90). Also such disco ntinuities were associated with increased permeabi lity t o c ol l oi d a l markers from the blood (88, 89), fu rther supporting the relationship between end othelial discon­ t i n ui t y and increa sed per meability. However, there i s also evidence f o r increa­ sed perm eabi lity through the a rt e r i a l endothelium with out r el a t i o n to any type of trauma. Thus, the penetration o f plasma p roteins through the a or t i c endothe­ lium of new-born rabbit s was la rger than tha t o f older animals (91). Therefore, the p o s s i bi l i t y should no t be disregarded that increased endothelial permeability f o r plasma constituents i n the ar te ries may also be o f physiological significan ce during growth, and possi bly also during repair o f a rt e r i a l tissue.

4.2.4 A r t er i a l tissue ground substance and perm eability

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be the sole mechanism o f t r a n s f e r o f l a b e l l e d cholestero l i n t o the normal a r ­ t e r i a l wal 1 .

5 . 1 . 3 Transfer o f l a b e l l e d cholestero l

An a l t e r n at i v e mechanism f o r the t r a ns f e r o f l a be l l e d chole sterol i n t o the a r t e ­ r i a l t i s su e may be physical exchange betw een serum l i p o p ro t e i n s and m embraneous l i p i d s o f the a r t e r i a l w a l l . Such ex change has bee n observe d i n the t r a n s f e r o f l a b e l l e d f r e e chole sterol between d i f f er e n t serum l i p o p r ot e i n classes ( 5 2 ) , and betw een serum l i p o p ro t ei n s and me mbraneous l i p i d s o f erythrocytes (108, 109) and t i s s u e c u l t u r e c e l l s (110, 111, 112). However, r a p i d t r a n s f e r o f l a ­ b e l l e d f r e e cholestero l by t h i s mechanism d oes n ot necessarily i n v o l v e actual net t r a ns f e r o f cholestero l (110, 111). Therefore, the existence o f actual t r a n s f e r o f plasma chole sterol i n t o the a r t e r i a l t i s su e has been questioned (25, 26, 102, 113). On the other hand, physical exchange react ions are r e s t r i c ­ ted t o l a be l l e d cholestero l i n the f r e e form (52, 5 3 ) , and consequently obser­ vations o f t r a n s f e r o f l a be l l e d c h o l e s t er y l ester i n t o the a r t e r i a l t i s su e ( I I I ) s t r on g l y i n d i c at e actual t r a ns f e r from plasm a. The exch ange o f l a b e l l e d f r e e cholestero l may occur duri ng r a p i d ly t r a n s i en t c o l l i s i o n s between d i f f e ­ r e n t l i po p r o t e i n complexes, and d i r e c t contact between the d if f e r e n t c h ol e s t e r ­ o l compartments probably i s a p r e r e q u i s i t e f o r i t ( 5 4 ) . Uptake o f l a b e l l e d chole sterol by t h i s mechanism w ould conseque ntly be r e s t r i c t e d t o the membrane­ ous l i p i d s o f endothelial c e l l s and c e l l s i n d i r e c t contac t w i t h these. There­ f o r e , the autoradiographic demonstration o f l a b e l l e d c h o l e s t e r o l , i n l a y er s o f the a o r t i c w a l l not i n contact w i t h endo thelial c e l l s ( 2 , 100, 114, 115) , con­ s t i t u t e s f u r t h e r evidence f o r actual t r a n s f e r o f chol esterol from pla sma i n t o the a r t e r i a l t i s s u e .

5 . 1. 4 Cholesterol biosynthesis

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the evaluation of such experime nts uncertain. Therefore, i t seems wise t o r e ­ f r a i n from qu antita ting the contributio ns of local synthesis and t r an s f e r ; from plasma, t o the chol esterol content i n the normal a rt e r i a l w a l l , at the present state o f knowledge.

5.2 The o r i gi n of excess ch oles terol i n ath erosclerotic lesions 5.2.1 Cholestero l content and atherogenesis

The f i r s t observations o f cholesterol and chol esteryl ester as cons tituents o f ath erosclerotic lesions date from as early as 1857(118). La ter, the demonstra­ t i on tha t cholesterol was i n f ac t the principa l l i p i d o f advanced ath eroscler­ o t i c les ions, and the observation of a correlat ion between coronary heart d i s ­ ease and seru m ch olesterol l e v e l s, stimulated more s pe cific i nt e r es t i n chol­ est erol and i t s functions i n atherogenesis (review and references: e.g . 119). Systematic studies on th e l i p i d composition o f human a r t e r ia l tissue with age and athero sclerosis have i ndicated that increased concentrations o f free and e st e r i fi e d chol esterol are characteristic features o f aging , and o f the form­ ation o f ath erosclerotic lesions (22, 120). Thus, i t now appe ars t o be f i r m l y established that the cholesterol content o f the a rt e r i a l wall increases during atherogenesis.

5.2.2 F i l t r a t i on o f serum lipo pro teins

Whereas the significance o f serum lip opro te in f i l t r a t i o n f o r cholesterol trans­ f e r i n t o the normal a r t e r i al wall i s doubtful, there i s much evidence f or such f i l t r a t i o n i n atherosclerotic a r t e r i a l tis sue. Thus, the presence o f serum l i p o ­ protein s i n atherosclerotic lesions has been demon strated w ith immunochemical (104, 121), immunohistochemical (93, 106, 107, 122, 123, 124), and e lectrop hor-e t i c (124) thor-echniquhor-es. Thhor-eshor-e invhor-estigations providhor-e strong support f o r thhor-e con­ cept o f lip opro tein f i l t r a t i o n as an important mec hanism of transf er o f cholest ­ erol i n t o atherosclerotic tissu e, but they do not provide any info rmation con­ cerning the mechanism o f cho lesterol dep osition.

5.2.3 Deposition o f cholesterol

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s tr a t i on that cho lesterol may accum ulate i n experimental ath erosclerotic lesions at serum choles terol levels below 50 mg/100 ml (V), provide strong evidence f o r the p ar t ic ip a t i o n o f other fac tors as w e ll . Other i nvestigato rs have suggested that deposition o f cholesterol may be due t o changes i n the aggregation state of acid glycosaminoglycans i n the a r t e r i al tissue (92). I n additio n, the forma­ t i on o f insolu ble complexes betw een acid glycosaminoglycans and serum lipo pro­ teins has been suggeste d t o be an i mportant mechanism f o r the deposition of cholesterol (93). On the other hand, r esults presented by Hollander e t a l . , i n ­ dicating p a r t i a l independence between dep osition o f lab elled cho lesterol and acid glycosaminoglycan metabolism (125), appear d i f f i c u l t t o combine w ith such a hy pothesis. I n addition, re sults presented i n V, ind icating that the deposi­ t i o n o f cholesterol i n experimental atheroscl erotic lesions i s rel ated t o i n ­ creased f i l t r a t i o n o f lab elled cholesterol from plasma, emphasize that not only the deposition but also the rate o f transfe r o f cho lesterol may be cha nged i n athero sclero tic lesions.

5.2.4 Increased f i l t r a t i o n o f serum lipo protei ns

The transfer o f lab elled cholest erol i n t o atheroscl erotic lesions i s consider­ ably higher than that i n t o normal a rt e r i a l tissue of the same experimental ani­ mal, as i ndicated by resu lts of V an d those o f other investigator s (e.g. 99, 125).

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I n ad dition, while the d is t r i b u ti o n of I - l ab e l l e d plasma proteins and H-cho l-esterol was dis sim ilar i n normal a o r t ic tissue (see above ), i t was si m ila r i n severely atherosclerotic a o r t i c tissue (83). This has been in ter preted as evidence f o r leakage of plasma li po proteins through the lumenal a r t e r i a l endo­ thelium i n t o the atherosclerotic tissue (83, 84) . This interpr et atio n gains sup­ port from our r e s u l t s, indica ting that d ra s t i ca l l y increased transfer o f l ab e l ­ led cho lesterol i n t o ath erosclerotic tissue (V) p a r al l el l ed excessive f i l t r a t i o n o f Evans blue-albumin complex ( I V ) . Therefore, i t appears probable tha t increa­ sed f i l t r a t i o n of plasma lipoproteins may play a s ig ni f i c an t r o l e i r i the depo­ s i t i o n o f cholesterol during atherogenesis.

5.2.5 Cholesterol biosynthesis

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i n experimental atherosclerotic lesions induced by mechan ical i n j ur y (V) . I n addition, the f i l t r a t i o n o f plasma p roteins was increased i n the lesions ( I V ) , and the presence o f plasma constituents may i n h i b i t cholesterol bios ynthesis, as suggested by i n v i t r o studies (127, 128). Therefore, i t does no t seem un­ reasonable t o suggest that chol esterol biosynthesis i s o f minor importance, a t lea st f o r the increments o f the cho lesterol content i n experimental atheroscle­ r ot i c lesions induced by mechanic al i n j u r y .

5.3 Cholesterol transfer i n a r t e r i a l tissue with i n t a c t endothelium 5.3.1 The r o le of cho lesterol i n normal a r t er i a l tissue

Cholesterol i s an impor tant constituent of membrane li poprotei ns i n a l l mamma­ l i a n c e l l s (54). The c hole sterol content may be of considerable significan ce f o r the normal str uctural and fun ctional properties of the c el l memmbrane. For example, decrease d c holesterol content i n the plasma me mbrane o f erythrocytes decreased t h e ir a b i l i t y to withstand osmotic s t r a i n (129, 130). No dou bt, ch ol­ est erol i s also important f o r the functional and st ru ctura l i n t e g r i t y o f the c el l s i n the a r t e ri a l w a l l . Furthermore, i t has also been sugges ted t hat inc re­ ased c holesterol content i s not always related t o athero sclero tic changes i n the a rt e r ia l wa l l , but may also f o r example r e f l e ct increments o f c e l l membrane material (27).

5.3.2 Cholesterol tra nsfer and metabolic a c t i v i t y

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fected. There fore, these studies do not contradic t the concept t hat the chol­ esterol content and tra nsfer o f plasma c holesterol may be depe ndent on the metabolic a c t i v i ty i n the a rt e r i a l w al l .

5.3.3 Active transport o f cho lesterol

On the basis o f his i n v i tr o studies, Jensen sugge sted th at transfer o f lab elled cholesterol from plasm a i n t o the a r t e r i a l tissue was du e t o ac tive transport inv olving primary, non energy-requiring binding o f serum lipo pro teins at the lumenal surface o f the endothelial c el l s (133) and subsequent h ydro lysis of e st e r i f i ed cho lesterol i n them (38). He suggest ed t ha t such tran sport might be mediated by pinoc ytotic a c t i v i t y (133). A l t er n a ti ve l y , by analogy wi th choleste­ r o l transport mechanisms i n tissue culture c e l ls , ac tive tran sport could also invo lve binding o f free and e s t e r i f i e d choleste rol at the c e l l membrane, ra pid hydrolysis of e s t er i f i e d chol esterol and i nt r a c e l l ul a r transport o f cho lesterol i n the free form (111, 128, 134, 135). Subsequently, chol esterol might be r e l e ­ ased from t he c e l l s , primarily i n the fre e form (111, 127, 128, 136). Both these me chanisms are consiste nt with the results of I I I which suggest a d i r ec t relation ship i n regions with i n t a c t endothelium bet ween f ree cholesterol radio­ a c t i v i t y and c holesteryl ester r a d i o a c t i v i t y, with a r a t i o between the fraction s of 20:1.

5.3.4 Control o f cho lesterol content

The c holester ol content i n the a r t e r i a l wall might conceivably be determined by both local biosynthesis and chole ster ol transfer from plasma . The in verse r el a ­ tionship between transfer o f lab elled fre e chol esterol and fre e cholesterol con­ tent ( I I I ) i n a or t i c tissue with i n t a c t endothelium suggest s an adjustment o f cholesterol transfer t o the local requirements i n the a r t e r i al w a l l . Werthessen and colla borators reported a simila r inverse relationship between ne t increase o f unlabelled cholesterol and i n i t i a l choleste rol conten t, from i n v i tr o studies on whole c a l f aortae perfused with a serum-containing med ium a t high pressure (200 mm mercury) (131, 136'). I n another study from th e same l abora tory, the data presented show a s imila r inverse rel ation ship (R= -0.54; p<0.05 calc ulated as Spe arman ' s rank corre lat ion c oe f f i c i e n t) a ft er perfusion of bovine aorta with a me dium contai ning serum (137). As suggested above, these r esults may be explained both by adjustment of the biosynthesis and by adjustment o f chol­ esterol transfer from serum lipo protei ns i n the medium. Howev er, the f ac t that

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t ut e strong evidence f o r Werthessen's hypothesis "that the a o r t i c content o f cholesterol i s subject t o co nt ro l" (136). Furthermore, the re su lts o f I I I ind icate that control o f the chol esterol content may invo lve adjustment o f chol­ esterol tra ns fer from plasm a i n response to the local requirements.

5.4 The c holesterol b a r r i e r

5.4.1 E ar l i e r evidence f o r a cholesterol ba rri er

Some in vestigato rs have h yp otheti cally postu lated the existence o f a b a r r i e r • against the i nf l u x and d eposition o f cho lesterol i n a rt e r i a l tissue (25, 132, 138)

I t has been suggeste d that the gradual break-down o f t hi s b a r r i e r during athero-genesis ma y a llow the accumulation o f serum choles terol i n the a rt e r i a l w a l l . This concept i s cons istent with a num ber of inter esting observations, f o r example the expo nentially increasing rat e o f chol esterol accumulation (139), and the ra pidly increasing i n f l u x o f lab elled cholesterol during the development of d i -e t a r i l y induc-ed at h-erosc l-erotic l-esi ons (discussion and r-e f-er-enc-es: 25, 138). 5.4.2 Endothelial i n t e g r i t y and f i l t r a t i o n o f serum lipo prot ei n cho lesterol As discussed i n d e t ai l above, there i s much evidence that increased f i l t r a t i o n of serum lipop roteins i s the most important mechanism f o r the accumulation o f chol esterol during atherogenesis. Such f i l t r a t i o n probably i s r es tr i c t ed t o a r t e r i a l tissu e with endothelium o f decreased i n t e g r i t y (see above). The ob ser­ vation o f increased tra nsfe r t)f labelled free and e s te r i f ie d cholesterol i n r e ­ gions o f both normal ( I I I ) and a therosclerotic (V) tissue having defective endo­ thelium indicates that the rat e of transf er o f cho lesterol by f i l t r a t i o n o f serum lipo pro teins may exc eed th at by a ctive transport even i n experimental animals w ith serum chol esterol levels below 50 mg/100 ml. I t may be in ferre d that i n individuals with higher serum chole sterol l e v e l s, t h i s diff erence would be even l a r g e r. Hence, i t may be suggest ed t hat the maintenance o f endothelial i n t e g r i t y might be a very important fac tor i n preventing excessive i nf l u x o f serum lipo prot ein cholesterol i nt o a rt e r i a l tissue.

5.4.3 Endothelia l i nt e gr i t y and d eposition of cholesterol

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d i f f e r e n t from t h a t i n plasma o f humans and experimental animals ( V I ; review and re ferences: 53)and i n more adv anced a t h e ro s c l e r o t i c lesio ns i n man (120, 149,

56 , 157). Mono-unsaturated c h o l e s t e r y l e s t e r f a t t y acids c o ns t i tu t e d the l a r ge s t f r a c t i o n i n the experimental lesio ns ( V I ) , f a t t y streaks and " f a t t y plaques ", whereas the f r a c t i o n w i t h di-unsaturated f a t t y acids was higher i n plasma ( V I ) and o ther types o f a t he r o s c l er o t i c l e s i o n s. I n a d d i t i o n, c h o le s t e r y l esters w i t h f a t t y acids containing three o r more double bon ds were m ore frequent i n the lesions than i n plasma ( V I ) . Increased am ounts o f such c h o l es t e ry l esters have also been obse rved by o ther i n v es t i g a t o r s i n a t h e r os c l e r o t i c lesions o f humans (146, 147, 150, 156, 158, 159). I t has been demo nstrated t h a t these es ters con­ t a i n f a t t y acids c h a r a c t e r i s t i c o f e s s en t i a l f a t t y acid d e f i ci e n c y (158). High content o f c h o l e s t e r y l esters w i th mono-unsaturated f a t t y acids may also be i n ­ duced by e s s e n t i a l f a t t y acid defic iency (160). However, the present r e s u l t s from experimental animal s without signs o f systemic e s s en t i a l f a t t y acid de­ f i c i e nc y i n d i c a t e t h a t , a t l e a s t i n t h i s study, t h i s f a c t o r does not induce the s p e ci f i c CEFA p a t t e r n observed. I t also seems u n l i k e l y t h a t l o c a l e s s en t i a l f a t ­ t y acid d e f i ci e n c y i s o f importance, as th e CEFA p a t t e r n i s s i m i l a r i n regions w i t h very d i f f e r e n t rates o f f i l t r a t i o n o f albumin ( V I ) and co nsequent d i f f e r e n t i n f l u x o f f r e e f a t t y acids from plasm a.

5 . 5 . 2 Local a r t e r i a l t i s su e f a c to r s

The CEFA p a t t e r n o f macroscopically normal i n t i ma from hu man ch anges w i t h age. I n c h i l d r en between 0 and 10 years o l d , c h o le s t e r y l o l e a t e was the predominating f r a c t i o n (150). However, d uring growth and aging t he CEFA composition i n the a r t e r i a l t i s su e grad ually became mo re s i m i l ar t o t h a t i n plasma. Thus, i n the i n t i m a o f humans over 30, the c h o le s t e r y l e s t e r composition wa s r a t h e r s i m i l a r t o t h a t i n plasma (120, 161). These observations support suggestions t h a t i n ­ f i l t r a t i o n o f the a r t e r i a l wall w i t h plasma c h o l e s t e r yl esters may c o n t r i b u t e very s i g n i f i c a n t l y t o the increase o f t o t a l c h o le s t er y l esters w i t h age. However, the c h a r ac t e r i s t i c CEFA p a tt e r n i n intima o f c h i l d r en suggests t h a t l o ca l mech­ anisms ma y als o be importan t i n determining the c h o le s t er y l e s t e r composition i n normal a r t e r i a l t i s s u e.

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The c h o l es t e ry l e s t e r composition wa s s i m i l a r , although the morphological d i s ­ t r i b u t i o n o f l i p i d s was d i f f e r e n t i n d i f f e r e n t regions o f the l e si o n ( V I ) . These r e s u l ts are not consi stent w i t h those o f other studies which demon strate s i g n i ­ f i c a n t l y d i f f e r e n t CEFA composition i n i n t r a c e l l u l a r and e x t r a c e l l u l a r l i p i d s (161, 162). The aberr ant r e s u l t s o f the present study ma y o f course be due t o species d i f f er e n c e s, as r a b b i t s were used i n the present s t u d y , whereas t he other studies have bee n performed on autopsy m a t e r i a l , derived from a d u l t o r e l d e r l y human populations . However, by analogy w i t h the r e s u l t s discussed above, they could conceivably also be r e l a t e d t o age d i f f e r e n c e s . Thus, i t may be spe­ culate d t h a t the i n t r a c e l l u l a r l i p i d composition m ay not change mu ch from t h a t during childho od, whereas the e xt r a c e l l u l a r c h o l e s te r y l e s t er composition m ay conform t o t h at i n plasma d uring growth and aging, not only i n normal t i ss u e , but also i n a t h e r o s c l er o t i c t i s s u e . However, the v a l i d i t y o f such an i n t e r p r e ­ t a t i o n i s d i f f i c u l t t o assess beca use o f the species d i f f e r e n c e s .

5 . 5 . 3 Choleste ryl e s t e r metabolism

Cholesteryl e s t e r metabolism i n the a r t e r i a l wall was studie d by isotope tech­ niques i n viv o ( V I ) . To our knowledge, such s tudies have not p r e v i o u sl y been performed w i t h normo-lipidemic experimental animals. The ev aluat ion o f the r e ­ s u l t s from these studies may be subje ct t o some l i m i t a t i o n s , beside s those d i s ­ cussed ab ove f o r a l l studies w i t h l a be l l e d c h ol e s t e r o l . As o nly one time i n t e r ­ val was s t u d i e d, i t was n o t possible t o e s ta b li s h when platea u values f o r the uptake o f r a d i o a c t i v i t y i n the d i f f e r e n t c h o le s t e r y l e s t e r f r a c t i o n s were reached. Therefore, the possible existence o f non-homogeneous c h o le s t e r y l e s t e r t i s s u e pools might l i m i t the v a l i d i t y o f the r e s u l t s . However, the observations were made a t a time p o i n t w e l l before the attainment o f e q ui l i b r i u m s p e ci f i c a c t i ­ v i t i e s between the t o t a l chole sterol pool i n plasma, and t h a t ( o r those) i n the a r t e r i a l t i s s u e. Results presented by Swell e t a l . i n d i c a t e t h a t t h i s holds t r u e also f o r d i f f e r e n t c h o le s t e r y l e s t e r s u b -f r a ct i o n s (163 ), a t l e a s t i n d i e t a r i l y induced a t h e ro s c l e r o s i s . F i n a l l y , we found t h at the c h o le s t e r y l e s t e r s p e c i f i c a c t i v i t i e s i n plasma exce eded the f i n a l s p e ci f i c a c t i v i t y i n the a r t e r i a l w a l l during the time studied ( V I ) , and t h a t the s p e c i f i c a c t i v i t i e s i n d i f f e r e n t c h o le s t er y l e s t e r s u b - f r a c ti o n s d i d not d i f f e r much ( V I ) . Therefore, d i f f er e n ce s i n s p e c i f i c a c t i v i t i e s o f d i f f e r e n t c h o l e s t er y l e s t e r sub- fract ions probably are r e l at e d t o diffe rences i n turn-over between the f r ac t i o n s .

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c a ti o n . Other i n v e st i g a t o r s have observ ed a decreased h y d r o l y s i s o f c h o l e s t e r y l o l ea t e , as com pared t o other c h o l e s t e r y l ester s (167, 1 6 8 ) , i n di c a t i n g a mec h­ anism o f s e l e c t i v e hydrolysis. F i n a l l y , i t i s conceivable t h a t interconversion o f other c h o l e s t e r y l esters t o c h o l e s t er y l o l ea t e , and m ore pro nounced r e t e n t i o n o f c h o le s t e r y l o l e at e than o f other c h o le s t e r y l ester s ( 3 7 ) , may be responsible f o r the increment o f t h i s p a r t i c u l a r c h o l e s t e r y l e s t e r s u b- f r a c t i on . Higher r a t e o f e s t e r i f i c a t i o n o f cholestero l w i t h o l e i c acid than w i t h other f a t t y acids i n the a r t e r i a l t i s s u e would gi ve higher s pe c i f i c a c t i v i t y i n t h i s f r a c ­ t i o n , as the f r e e chol esterol s p e c i f i c a c t i v i t y was higher than the c h o l e s t e r y l e s t e r s p e ci f i c a c t i v i t y . On the other hand, the other mechanisms discussed abo ve would g ive lower s p e c i f i c a c t i v i t y i n c h o l e s t e r y l oleate than i n the other cho­ l e s t er y l e s t e r s u b - f r a c t i o n s . Therefore, the absence o f cons istent differenc es i n s pe c i f i c a c t i v i t i e s , between the three main, more satu rated c h ol e s t e r y l e s t e r s u b - f r ac t i o n s , may i n d i c a t e t h at no s i n gl e mechanism i s responsible f o r the c h a r a c t er i s t i c c h o le s t e r y l e s t e r composition o f the l e si o n s . I t may also support suggestions t h a t both s e l ec t i ve h y d r o l y s is and s e l ec t i ve e s t e r i f i c a t i o n may be s i g n i f i c a n t f o r the c h o le s t er y l e s t e r composition i n a t h e r o s c l er o t ic lesions (169). The s p e c i f i c a c t i v i t y o f the three main, more saturat ed c h o le s t er y l e s t e r

f r a c t i o n s was considerably higher than t h a t o f c h o l e s t e r y l ester s w i th f a t t y acids containing three o r more double bon ds ( V I ) , suggesting a low tur nover o f the l a t t e r . This suggestion i s supported by the observation t h at the r e l a t i v e concentration o f such c h o l e s t e r y l esters increased, a f t e r removal o f chole ste­ r o l during the r e s t o r a t io n o f the a r t e r i a l w a l l s t r u c t u r e ( V I ) . P r ef e r en t i a l depos ition o f these s p e c i fi c c h o l e s t e r y l esters may r e f l e c t a p r o t e c t i v e response against the sclerogeni c e f f e c t o f other forms o f c h o l es t e r o l, as hy pothetized by Abdullah e t a l . f r o m observations on the e f f e c t o f d i f f er e n t forms o f choles­ t e r o l i n subcutaneous' t i s s u e implants i n r a t s (170).

5.6 Cholesterol removal and at herosclerosis

5 . 6 .1 Decreased c holesterol removal and a therosclerosis

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5 . 6 . 2 Regression o f a t h e r o s c l er o t i c changes

I n the experimental a t h e r o s c l er o t i c lesions i n v es t i g a t e d i n the present s t u d y , the cholestero l concentration i n regions which we re s t i l l devoid o f endothel­ i a l l i n i n g was 3 times as hig h as i n r e - en d o t h e li a l i ze d regions ( V ) . This d i s ­ crepancy betw een d i f f e r e n t regions which had a l l been de nuded o f e n d ot h e l i a l l i n i n g by the i n i t i a l trauma, i n d i c at e s t h a t chole sterol i s r e a di l y eliminated during r e pa i r and r e - e nd o t h e l i a li z a t i o n o f the l e s i o n . I n the non- endo thelial-ized r e g i o n s , the discrepancy i n uptake o f l a b e l l ed f r e e and e s t e r i f i e d c h o l e s t ­ e r ol may sugg est t h a t f a r more cho lesterol enters the a r t e r i a l wall than i s de­ posit ed i n i t ( V ) . This suggestion i s supporte d by the f a c t t h a t , during the 4 weeks f o l l o w i n g the trauma, ca. 10 mg/g dry t i s s u e weight o f chole sterol has been deposited i n the YARD, wher eas the uptake o f r a d i o a c t i v i t y i n the same r e ­ gions suggests t h a t 15 mg m ay ha ve pas sed through the l e s i o n during the 24 hours o f exposure t o r a d i o a c t i v e chole sterol ( V ) . The low r a t e o f d e p o s i t i o n , i n s p i t e o f the r a p i d t r a n s f e r o f c h o l e st e r o l , may be r e l a t e d t o high capaci ty f o r remo­ val o f chole sterol i n a t he r os c l e r o t i c t i s s u e o f normo-1 i p i demi c r a b b i t s ( V ) . The que stion, whether d i e t a r i l y induced experimental a t he r os c l e r o t i c l e s i o n s may regress o r n o t , has been the subject o f some contr oversy. Some i n v e s ti g a t o r s have bee n unable t o show an y change i n the lesions a f t e r withdrawal o f a c h o l ­ e s t e r ol - r i c h d i e t (171). Others have observe d t h a t a t he r o s c l er o t i c changes de­ creased a f t e r the cessation o f chole sterol feedin g, while on the other hand the cholestero l content i n the a r t e r i a l wall d i d not change (172). F i n a l l y , some i n v e s t i ga t o r s have bee n able t o demonstrate t h a t the incidence o f a t h e r o s c l er o t ic changes, as well as th e concentration o f cholestero l i n the a r t e r i a l w a l l de­ creased s ig n i f i c an t l y a f t e r long periods on c h o l e s t er o l - f r e e d i e t s (173). Unpub­ l i s h e d r e s u l t s from our l a b o r a t o ry i n d i c a t e t h a t regress o f experimental athero­ s c l e r o t i c lesio ns induced by mech anical trauma i n normo-lipidemic r a b b i t s , was r e l at e d t o the r e s t o r a t i o n o f an i n t a c t endothelial l i n i n g ( 3 4 ) . I n a d d i t i o n, the present r e s u l t s suggest t h at cholestero l may be elimin ated during re-endo­ t h e l i a l i z a t i o n o f a t he r os c l e r o t i c t i s s u e ( V ) . Therefore e n d o t he l i a l i n t e g r i t y appears t o be an imp ortant f ac t o r , not only f o r the mainten ance o f the normal cholestero l content i n the a r t e r i a l w a l l , but also f o r the removal o f c h ol e s t ­ e r ol deposits during regression o f a t he r o s c l er o t i c changes.

5 . 6 . 3 Cholesterol removal-possible mechanisms

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36

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6, ENDOTHELIAL INTEGRITY AND EXPERIMENTAL ATHEROSCLEROSIS

6.1 Experimental atherosclerosis induced by mechanic al trauma

The develo pment of l i p i d - r i c h a r t e r i a l lesions was rec ent ly reported following certa in types o f defined mechanical i nj u r y i n normo-lipidemic rab bits (32) and ra ts (183) These o bservations ind icate that certain types o f mechanical i n j ur y may i n c it e tissue responses re lat ed t o those i n atherosclero sis (33 ), and, theref ore, must be considered as one pos sible i n i t i a t i n g fac tor i n atherogenesis. I t was sugges ted t hat the char acter istic accumulation o f l i p i ds i n atherosc lerosis i s related t o defective endothelium (33). This suggestion receives strong support from th e resu lts of V, indicating that deposition o f cholesterol was confine d to regions with defective endothelium i n experimental ath erosclerotic lesions induced by superficial mechanical i n j ur y with larg e area. The absence o f endo­ t h e l i a l l i ni n g i n these r egions may allow the i nf l u x of plasma lipopro te in ch ol­ est erol i n quantities exceeding the capac ity f o r removal, leading t o deposition o f cholesterol .

6.2. Di e t a r i l y induced experimental atherosclerosis

Not only experimental mechanical i n j u r y , but also pre-ex ist ing endothelial de­ fects i n regions subject t o increased hemodynamic str ess, may be re lated t o the l oc a l i z a ti o n o f atheroscl erotic lesion s. Thus, a f t e r the induction of hyperchol­ esterolemia, the incidence o f such lesi ons was increased i n region s, d i s tr i b u t e d s i m i l a r i l y as regions with defective endothelium i n the normal rabb it ( 8 , see I I and above). However, atherosclerotic lesions with l i p i d deposition develop i n these areas on ly i f the blood l i p i d levels o f the experimental animals are inc rea­ s e d ^ ) , but at very low levels i n areas w ith l ar g e r , experimentally induced endo­ t h e l i a l defects (32). Therefore, i t may be hypothetiz ed that changes ei ther i n blood l i p i d levels or i n endothelial i n t eg r i t y , as w ell as conc urrent changes i n both f ac to rs , may be s i gn if i c a nt f or the formation o f ath erosclerotic lesions with deposition of l i p i d s .

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7 , A B S T R A C T

S t r uc t u r a l and f u n c t i o n a l p r o pe r t i e s o f the a o r t i c endothelium, and l o c a l t r a n s ­ f e r mechanisms f o r f r e e and e s t e r i f i e d chole sterol i n normal aorta and i n a o r t i c experimental a t h e r o s c l er o t ic lesio ns have bee n s tudied. For the i n v es t i g a t i o n s i t was necessary t o develop new techniques f o r the study o f endo thelial c e l l v i a b i l i t y and s t r u ct u r e ( I I ) and a method f o r the u l t r a -m i c r o assay o f c h o l ­ e s t e r o l i n t i s s u e ( I ) . These techiques have been com bined, and tog ether w i th isotope techiques applied t o normal aorta and experimental a t h e r os c l er o t i c lesions induced by s u p e r f i c i a l mechanical i n j u r y w i t h l a r g e area i n normo-1 i p i demi c r a b b i t s .

The endothelium o f the normal a o r t i c surface was s t r u c t u r a l l y and f u n c t i o n a l ly heterogeneous ( I I ) . Defective endothelium w as present where increased hemodyna­ mic s t r a i n occurs ( I I ) . The increased in cidence o f a t h e r o s c l er o t i c changes i n the same regions sugg ests t h a t decreased end othel ial i n t e g r i t y may be an imp ort­ ant f a c t o r i n the i n t e r r el a t i o ns h i p between he modynamic s t r a i n and a t h e ro s c l e r ­ o s i s . Mural microthrombi were observ ed i n regions w i t h d e f e c t i ve endothelium ( I I ) , suggesting t h a t decreased e ndothelial i n t e g r i t y may also be r e l a t e d t o throm botization.

Structural heterogeneity was p a r a l l e l l e d by hetero geneous permeability charac­ t e r i s t i c s ( I I , I I I ) . The r e s u l t s i n d i c a t e t h a t f i l t r a t i o n o f plasma p r o t ei n s probably i s confined t o regions w i t h d e f ec t i v e endothelium ( I I I , I V , V). I n normal a r t e r i a l t i s su e covered w i t h i n t a c t endothelium, an inver se r e l a t i o n ­ ship was observed bet ween cho lesterol content and t r a n s f e r o f l a b e l l e d c h ol e s t ­ e r o l from pla sma ( I I I ) , suggesting an adjustment o f cholestero l t r a n s f e r t o l o c a l requirements o f cholestero l i n d i f f e r e n t regions o f the normal a o r t i c t i s s u e. I n a d d i t i o n, a d i r e c t r e l a t i o n s h ip was found betw een the t r a n s fe r o f l a be l l e d f r e e cholestero l and t h a t o f l a b e l l e d e s t e r i f i e d c h o l es t e r o l, w i t h a r a t i o between the f r ac t i o n s o f 20:1 ( I I I ) . This i s consistent w i t h the concept o f a c t i v e t r a n s p o rt o f c h o l es t e r o l, i n vo l v i n g r a p i d hydrolysis o f c h o l e s t e r y l e s t e r , a me chanism su gges ted by others.

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I wish to express m y g ratitude to :

Professor Holger Hydén, the prefect of the Department of Histology, where l a ­ boratory f ac i l i t i e s fo r the Ge riatric Research group, sponsored by the Swedish Medical Research C ouncil, have bee n p rovided.

Professor Lars Werkö for his intere st and support throughout the studies sum­ marized i n the present thes is.

Dr. Sören Bj örkerud, my s cie nti fic teacher and my coll aborator throughout the study, for generous support and con structive c ritic ism.

Mrs. Ingeli Andreasson, Mrs. Lena Hjalmarsson, Mrs. Gun Olsson and Miss Ann-Marie Wasshede fo r excellent technical assistance.

Miss Gull Grönstedt fo r careful secretarial work.

Dr. Irwin Levitan fo r c r it i c al reading and cor rection of the manuscript. Dr. Tore Scherstën for valuable cr it icism of the manuscript.

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