Revised concepts of athero-genesis

A review

More than 20 years ago Page1 r epo r t ed on the supposed causes of atherosclerosis. Most of the investigators in this field a re no longer "new and

Keith G. McCullagh, Ph .D. , <i u i t e shiny." They have toiled long and dili-B.V.Sc. M.R C.V.S. gently a n d communica ted with each o ther pro-

fusely, and yet "cures" for atherosclerosis have Department of Pathology, University of i - j ^ t ^ - n Bristol England n ° e e n proclaimed at least twice a year as Page a n d ' had expec ted . T h a t is not to say that knowledge Division of Research has not advanced in the past 2 decades. T h e

recognit ion of the ma jo r impor tance of this dis-ease process has at t racted many new investiga-tors. Publications have been prolific and newer technology has a d d e d sophistication to old ex-per iments . Yet few advances have been of suffi-cient magn i tude to excite the at tention of the clinician, su rgeon , or pathologist with o ther areas of interest . Ins tead , advances in unde r -s tanding atherosclerosis have been subtle and complex, as is the disease itself.

T h e r e are several reasons why this should be so. For one, arteries tend to react to a wide variety of in jur ious or in f l ammatory stimuli with a ra ther u n i f o r m set of responses , so that divin-ing the na tu re of the st imulus f r o m an examina-tion of the lesion, always a hazardous process for the pathologist , no longer has any credibility in degenerat ive arterial disease. For ano the r ,

247

248 Cleveland Clinic Quar ter ly Vol. 43, No. 3

atherosclerosis is a disease which many assume takes a lifetime to de-velop to the stage where clinically sig-nificant occlusion of an artery occurs (Fig. 1). Dur ing that l ifetime, the ar-tery may have been subjected to many stimuli. Even if a full history of these influences were available, at-tempts to d i f ferent ia te in jur ious stim-uli f r o m those which are relatively harmless would be speculative at best. So investigators have relied on exper imenta l work, of ten of only a few minutes , weeks or months , to f ind keys to h u m a n problems which may have developed over much longer periods of t ime. T h i r d , ath-erosclerosis is commonly held to be a multifactorial disease. If the re is a "mosaic" theory of hyper tens ion 2 and many potential "causes" of neopla-

sia,3 then atherosclerosis can surely claim to have a risk for all seasons. At the last count there were at least 35 documen ted epidemiologic factors which predispose towards athero-sclerotic hear t disease.4 T h e com-plexity of the interrelat ionships be-tween these factors and at l ierogene-sis seems limited otdy by our own h u m a n need to simplify epidemiol-ogy in o r d e r to c o m p r e h e n d it.

One simplification which many have made is to grade risk factors on the s t rength of their pr imary associa-tions, and to ignore factors whose sta-tistical associations with enhanced ar-terial disease are small. T h e separa-tion of major f rom minor risk factors in this way is rational because it can-not be said with certainty that the correlations between atherosclerotic

F i g . 1. F i b r o u s s t enos i s o f a n e x t r a m u r a l h u m a n c o r o n a r y a r t e r y in a p a t i e n t w h o d i e d with m y o c a r d i a l i n f a r c t i o n . N o r m a l v a s c u l a r a r c h i t e c t u r e has b e e n d e s t r o y e d a n d r e p l a c e d by d e n s e f i b r o u s c o n n e c t i v e t i s sue . A r e a s of ca lc i f i ca t ion a n d l y m p h o c y t i c i n f i l t r a t i o n a r e a lso visible ( h e m a -toxyl in a n d e o s i n s t a i n . X32).

Winter 1976

disease and the latter have any patho-genetic meaning. On the other hand , those factors whose associations with atherosclerotic disease are strongest are unlikely to be exerting their influ-ence indirectly. Presumably, they are related to the progression of the dis-ease by virtue of some casual relation-ship to it.

Epidemiologic studies

Much of this graded classification of risk has come about th rough tedi-ous but of ten highly successful epide-miologic studies. Of these, the most noteworthy is the Framingham Study,5 which since 1949 has assem-bled and moni tored a cohort of 2,176 men and 2,669 women. T h e results f r o m Framingham unequivocally demonstra te that the risk of suffer ing a major atherosclerotic event (coro-nary heart disease, a therothrombotic brain infarction or intermit tent clau-dication) is strongly related to age, blood pressure, serum cholesterol, cigarette smoking, relative weight, glucose intolerance, and an electro-cardiogram indicative of left ventric-ular hyper t rophy. T h e relationships between these risk factors and the development of atherosclerotic dis-ease are such that single and multiple regression equations can be calcu-lated which describe the incidence in terms of these factors. T h e highly significant regression coefficients im-ply that the average populat ion inci-dence is dependent u p o n the levels of these risk factors. This is as close to a demonstrat ion of etiologic connec-tion between these factors and ather-osclerotic disease as any epidemio-logic study is capable of achieving.

A fascinating aspect of the data f rom Framingham is that the risk of suffer ing a fatal coronary attack can

Revised concepts of atherogenesis 249

be calculated on a personal level. T h u s a 50-year-old man who smokes cigarettes, has a normal glucose toler-ance and electrocardiogram, a sys-tolic blood pressure of 135 mm Hg, and a serum cholesterol of 235 mg/dl , has a probability of 23/1000 of devel-oping coronary heart disease within 2 years and a probability of 108/1000 within 8 years.5 This 10.8% chance would be decreased by almost 4% if he did not smoke cigarettes and con-versely, it would be increased by a similar amount if his serum choles-terol level was 50 mg/dl higher .

Of course, the population of Fra-mingham, Massachusetts is not nec-essarily that of Cleveland, Ohio or Bristol, England, and caution must be exercised in extrapolat ing the Fra-mingham data to the rest of the United States or to other countries. Similar care should be used in evalu-ating other local prospective studies, and yet substantial agreement on the major coronary risk factors has been achieved th rough national6,7 and in-ternational8 cooperative studies of cardiovascular disease. First, athero-sclerosis is a disease which dramati-cally increases in severity with age and tends to be more severe in men than in women of the same age. These two primary associations are probably crucial in fully unders tand-ing the disease, yet of less immediate concern to the patient, since he or she can do nothing to alter them. If we accept for the moment the pres-ence of these two pr imary associa-tions, the most important of the re-maining risk factors for coronary hear t disease can be listed as (not nec-essarily in order of importance): (1) hyperl ipoproteinemia, (2) hyperten-sion, (3) cigarette smoking, and (4) impaired glucose tolerance.

250 Cleveland Clinic Quarterly

This assumes that o ther factors such as obesity and left ventricular hyper t rophy are impor tant by virtue of their relationship to one of these four , which admittedly may not be the case. But few would doubt the likelihood of a rapid acceleration of coronary hear t disease in the pres-ence of these four factors.

T h e major risk factors cited consti-tute a primary f ramework within which any modern theory of athero-genesis must be constructed. It is no longer sufficient to suggest that oc-clusive atherosclerosis develops in some and not others by a r a n d o m lottery. What has evolved f r o m the epidemiologic work is that there are now clear-cut circumstances u n d e r which we know accelerated athero-genesis occurs. It is extremely impor-tant that a modern theory of athero-genesis before all else should postu-late a mechanism which explains these relationships.

In recent years a considerable amount of new informat ion has been obtained about fundamen ta l aspects of arterial behaviour. It is worthwhile to consider whether such new infor-mation brings us any closer to defin-ing the exact causes of atherosclero-sis. Much of this recent evidence has subtly altered mechanistic concepts, so that it is now possible to state that none of the classic pathways of ather-ogenesis (lipoprotein infiltration the-ory, thrombogenic hypothesis and in-jury-repair concept) is sufficient on its own to answer the question of how atherosclerosis is caused. What is needed is a unified theory which can rationally incorporate the relevant parts of older theories.

Changes in ou r concepts of a thero-genesis in the past 2 decades are best illustrated by re ference to specific

Vol. 43, No. 3

problems. T h e discussion below fo-cuses on certain impor tant facets of atherogenesis which I believe must be unders tood and incorporated in any unifying theory of atherosclerosis.

The entry of lipoproteins and lipids into the arterial intima

It is axiomatic that there is an accu-mulation of lipid in atherosclerotic lesions. In practice, the extent of this lipid accumulation varies with the na-ture of the plaque, being severe in fatty streaks and f rank a the roma and less so in fibrous plaques. It is not helpful to characterize all athero-sclerotic lesions as fatty, for there are many cases of severe intimal fibrosis where virtually no lipid can be dem-onstrated histologically within the le-sion (Fig. 1). The re are also fibrous plaques which contain large masses of amorphous lipid at their centers (Fig. 2). T h e distinction between these d i f fe ren t forms of the athero-sclerotic lesion and the a m o u n t and quality of lipid which they contain has been painstakingly presented by Smith et al.9"12 Some of the results f r o m their laboratory studies are pre-sented in Figure 3. T h e absence of morphologically recognizable lipid f rom many human fibrous plaques is mir rored by a cholesterol concentra-tion in the lesion no d i f fe ren t f r o m that of normal intima. Of those le-sions which can be regarded as fatty, there are two quite distinct types, fatty streaks in which most of the lipid is intracellular, and fibrofatty plaques in which most of the lipid is in amorphous extracellular pools. A remarkable f inding of Smith et al, amply confi rmed by others , is that the lipid in foam cells in fatty streaks has a bizarre fatty acid composition unlike that of plasma, whereas the

Winter 1976 Revised concepts of a therogenesis 251

F i g . 2 . H u m a n c o r o n a r y a r t e r y . C o m p l e x a t h e r o s c l e r o s i s . A l a r g e p o o l of a m o r p h o u s a n d crys ta l -l ine l ipid is p r e s e n t in t h e t h i c k e n e d i n t i m a a n d is s u r r o u n d e d by h y a l i n e c o n n e c t i v e t i s sue . T h e tail o f a n o b d u r a t i n g t h r o m b u s is p r e s e n t in t h e l u m e n ( h e m a t o x y l i n a n d eos in s t a i n . X52).

extracellular lipid f r o m fibrous plaques has similarities to that of plasma ^- l ipoprote in . This d i f fer -ence is exemplif ied in Figure 3 by the highly unusual oleate:linoleate (18:1/ 18:2) ratio in the cholesteryl esters of fatty streaks. This suggests that the

lipid in some of the most cellular le-sions is derived f r o m local synthesis, or at least that plasma lipid is subject to sizeable metabolic alteration or se-lective inf lux. Put ano the r way, lipid accumulat ion in fatty streaks appear s to be a result of atherosclerotic

252 Cleveland Clinic Quar ter ly Vol. 43, No. 3

HUMAN ATHEROSCLEROSIS IOata from Smith, ret.34)

Normal intima (adulti Fatty streak

I C 6.0 EC 4.6 18:1/18:2 0.73

TC 31.5 EC 27.5 18:1/18:2 4.33

Fibrous plaque

0.59

Fibrous plaque with amorphous center

Amorphous lipid

0.68

EXPERIMENTAL CANINE ATHEROSCLEROSIS (Data from Butkus et al, réf.351

Normal artery Fibro-fatty plaque

TC 1.4 EC 0.3 18:1/18:2 0.39

TC 17.8 EC 14.5 18:1/18:2 4.02

SPONTANEOUS ELEPHANT ATHEROSCLEROSIS (Data from McCullagh, ref. 107)

Normal intima & media Fibrous olaoue

TC 1.6 EC 0.8 18:1/18:2 2 .10

TC 3.5

" - ^ • V r - T i EC 2.6 •., 18:1/18:2 4.34

F i g . 3 . D i a g r a m m a t i c r e p r e s e n t a t i o n of t h e typical a m o u n t s o f c h o l e s t e r o l a n d its e s t e r s p r e s e n t wi th in d i f f e r e n t t ypes o f a t h e r o s c l e r o t i c l e s ion . T h e d a t a h a v e b e e n se lec ted f r o m t h e s o u r c e s s h o w n to i l l u s t r a t e t h e u n i q u e c h a r a c t e r i s t i c s o f t h e h u m a n f i b r o u s p l a q u e . L ip id a n d c h o l e s t e r o l a r e s h o w n in b l a c k . T C —tota l c h o l e s t e r o l c o n c e n t r a t i o n in m g / 1 0 0 m g d r y t i s sue ; EC — e s t e r i f i e d c h o l e s t e r o l c o n c e n t r a t i o n in m g / 1 0 0 m g d r y t i s sue ; 18 :1 /18:2-weight r a t i o o f c h o l e s t e r y l o l e a t e t o c h o l e s t e r y l l i n o l e a t e .

change ra ther than a cause of it. quantities of cholesterol in the amor-It will be seen f rom the data in phous centers of "a the romatous"

Figure 3 that a l though there are large plaques, the cholesteryl oleate-lino-

Winter 1976 Revised concepts of a therogenesis 253

leate ratio is similar to that of normal intima and dissimilar to that of fatty streak. This argues strongly in favor of a d i f f e ren t pathogenesis fo r the two types of lesions. Unfor tuna te ly , the issue has become confused by analysis of exper imenta l lesions (illus-trated in Figure 3) by r e fe rence to a morphologically impressive, induced atherosclerosis in dogs,1 3 which have invariably revealed cholesteryl ester fatty acid pat terns similar to those of h u m a n fatty streak. It is t he re fo re possible that the morphologic simi-larity of many such exper imenta l le-sions to advanced h u m a n atheroscle-rosis is for tu i tous and that they may not provide particularly accurate models of lipid metabolism in h u m a n f ibrous plaque and a the romatous le-sions. Nei ther are spontaneous ani-mal lesions, occurr ing in the absence of hyper l ip idemia, reliable indicators of the processes involved in lipid ac-cumulat ion in h u m a n f ibrous plaques. T h e e lephant plaques illus-t ra ted diagrammatically in Figure 3 display a r a the r bizarre fatty acid pat-tern which at first sight seems incon-sistent with their o r d e r e d and rela-tively acellular s t ructure .

One of the p redominan t lipids to accumulate in h u m a n , exper imenta l and spontaneous mammal ian athero-sclerotic lesions is cholesteryl oleate and in exper imenta l animals at least, most of this ester accumulates f r o m plasma.1 4 Kinetic studies of nonester-ified cholesterol in a the romata have also shown that despite the presence of local cholesterol synthesis,15 the bulk of the lesion nonester i f ied cho-lesterol is derived f r o m plasma.1 6

T h e impor tan t question which bears on the pathogenesis of atherosclero-sis is how does this inf lux of lipid occur? Is it carr ied into the intima as part of intact l ipoprotein molecules,

or is there a t rans fe r of lipid f r o m plasma l ipoprotein to arterial cell in-d e p e n d e n t of l ipoprotein f lux .

T h e work which suppor t s the lipo-protein infi l trat ion theory by provid-ing evidence for the presence of se-r u m l ipoproteins in a the romatous tis-sues is primari ly histochemical. Uti-lizing f luorescent antibodies of vary-ing puri ty , Watts,17 Kao and Wis-sler,18 Woolf and Pilkington,1 9 Wal-ton and Williamson20 and Hof f et al21

have demons t ra t ed material immu-nologically identical to s e rum /^lipo-protein in atherosclerotic lesions. T h e specificity of the reaction is a crucial issue here , fo r insufficiently pur i f ied f luorescent ant ibody may cross react with smooth muscle mate-rial. However , if we assume that the identification of /3-lipoprotein de-rived material by this technique is correct , it would a p p e a r that al-though /3-lipoprotein can be detected in atherosclerotic lesions there is no clear association between the a m o u n t of l ipoprotein and the severity of the lesion. In addi t ion , f luorescence is not normally seen in undiseased ar-teries. Similar conclusions are reached by immunoe lec t rophore t i c studies of l ipoproteins extracted f r o m the intima.2 2 - 2 4

T h e situation can be summar ized by saying that the bulk of the lesion cholesterol appea r s to be derived f r o m plasma, and that some but not all of this cholesterol can be re-ex-tracted f r o m the arterial wall in the f o r m of l ipoprotein molecules which are similar in flotation and immuno-logic characteristic to the low density l ipoproteins of plasma. It is t empt ing to regard the mat ter of l ipoprotein inf lux as proven, but we cannot say to what extent entry of intact l ipopro-tein contr ibutes to the bu i ldup of the sequestered a the roma lipid. We

254 Cleveland Clinic Quarter ly Vol. 43, No. 3

know f r o m other studies that lipo-protein lipid, dissociated f r o m the apoprote in , may be taken u p by ath-erosclerotic intima and it may be that this is quantitatively a more impor-tant process than infiltration of entire l ipoprotein molecules.

Endothelial permeability and endo-thelial damage

T h e endothel ium lining the aorta and its major branches, including the coronary arteries, normally acts as a barr ier to the passage of proteins and other large molecules. T h e cells of the endothel ium are separated f r o m one another by tortuous intercellular channels that are too small to allow the passage of any but the smallest of plasma proteins.2 5 Recent observa-tions with the electron microscope on the passage of ul trastructural marker proteins have shown that this barr ier is made more impermeable by the presence of complete tight junct ions

in cerebral arteries26 and by partial or incomplete tight junct ions in other large arteries.27 Extensive membrane appositions, or gap junct ions have also recently been described in aortic endothelium.2 7 '2 8 Where a gap is present between opposed cell mem-branes in these regions it appears to be only 20 to 40Â wide. It is quite irrational to propose çhat unde r nor-mal physiologic conditions molecules of the size of low density lipoprotein could pass into the subendothel ium by this route. This is emphasized by Figure 4 which is a proport ional scale diagram illustrating the relative sizes of gaps and particles.

T h e r e is evidence which suggests that endothelial junct ions are some-what labile and that unde r conditions of hypertension2 9 there may be a re-versible opening of tight junctions without cell separat ion. T h e opening appears to be sufficient to allow the passage of horseradish peroxidase

Chy lomicrons Pre-yS-l ipoprotein

SUBENDOTHELIUM

F i g . 4 . D i a g r a m m a t i c r e p r e s e n t a t i o n of n o r m a l a r t e r i a l e n d o t h e l i u m d r a w n to a scale o f 25 n m (250 A) = 1 m m . T h e d r a w i n g s h o w s n o r m a l i n t e r c e l l u l a r c h a n n e l s c o n t a i n i n g foca l t i g h t j u n c t i o n s a n d e x t e n s i v e g a p j u n c t i o n s . P l a s m a l e m m a l vesicles v a r y f r o m 600 to 1000 A in d i a m e t e r , b u t t h e n e c k o p e n i n g o f s u r f a c e vesicles is r a r e l y l a r g e r t h a n 300 A . P l a s m a l i p o p r o t e i n pa r t i c l e s a r e d r a w n to a s imi l a r sca le . T h e e n d o t h e l i a l cell n u c l e u s , m i t o c h o n d r i a a n d o t h e r o r g a n e l l e s h a v e b e e n o m i t t e d f o r c la r i ty . T h e b a s e m e n t m e m b r a n e - l i k e m a t e r i a l o f t e n s e e n in t h e n e c k o f m e m b r a n e a t t a c h e d vesicles is a lso n o t s h o w n . T h e d r a w i n g is e q u i v a l e n t to a n e l e c t r o n m i c r o g r a p h o f m a g n i f i c a t i o n X40,000 .

Winter 1976 Revised concepts of a therogenesis 255

(mol wt 40,000) but not ferr i t in (mol wt 500,000). Such physiological labil-ity does not appea r to be sufficient to allow passage of plasma low density l ipoprotein (mol wt 3,000,000). How-ever, it has recently been claimed8 0

that tight junct ions in arterial endo-thel ium are reversibly dilated by physiologic concentrat ions of angi-otensin, and that the increase in permeabili ty is sufficient to allow t ranspor t of l ipoprotein. Increases in pore size of this magni tude have not been demons t ra ted unequivocally in normal cont inuous endo the l ium, and this view is not in ag reement with o ther evidence.2 9 For the t ime be ing it is fair to conclude that in te rendo-thelial passage of l ipoprote in is un-likely to occur provided the endo-thelial cells are u n d a m a g e d .

T h e r e is ano ther route by which large molecules may traverse the en-do the l ium, and that is within the plasmalemmal vesicles that are con-tinually fo rming f r o m the cell sur-face, travelling randomly in the cell in ter ior , and re fus ing with the mem-b r a n e ei ther on the same or ano the r side of the cell (Fig. 4). These vesicles are abou t 700 A in diameter 3 1 and could, the re fo re , accommodate nor-mal low density l ipoproteins which have molecular diameters of 170 to 260 A.32 It is accepted that this "large pore system" can act as a rou te of t ransendothel ia l passage of plasma prote ins , but with much smaller mol-ecules than plasma low density lipo-protein there is considerable molecu-lar sieving.31 It may be that quant i ta-tively this route allows the entry of only insignificant amounts of lipo-prote in , but the exper iments which would enable us to make such a state-ment with conf idence have not been p e r f o r m e d .

A fair appraisal of the recent work on arterial endo the l ium and l ipopro-tein t ranspor t would be that u n d e r normal conditions the endo the l ium acts as an incomplete bu t pret ty effec-tive barr ier to the passage of plasma l ipoproteins. Rather than stress that l ipoproteins normally percolate t h rough the arterial wall f r o m lumen to lymph vessel, it would be better to emphasize the relative impermeability of normal intact endo the l ium.

But, as we have seen, many athero-sclerotic lesions, both h u m a n and an-imal, contain large amoun t s of low density l ipoprotein which appear s to be indistinguishable f r o m that of plasma. If it is assumed that the ma-jori ty of this l ipoprotein is derived directly f r o m plasma, it is obviously necessary to postulate that the endo-thelial barr ier over the plaque is or has been breached . Until recently the morphologic evidence for this has been equivocal. Endothel ia l cells sur-moun t ing plaques are of ten abnor-mal in being highly vacuolated and possessing cytoplasmic alterations, 33,34 k u t f r a n k changes in intercellular gaps have been difficult to demon-strate. However , recent examinat ion of "en face" prepara t ions of arterial endo the l ium by light microscopy35

and scanning electron microscopy36

have revealed distortion and loss of endothelial cells overlying intimai plaques. In addi t ion, many investiga-tors have shown that endothel ial loss or damage can result f r o m a wide variety of chemical, physical, and im-munologic injuries . Such results need be as minor as a local variation in blood flow or the presence of a circu-latory toxin such as carbon monox-ide.

T h e loss of endothel ial integrity appear s to be the c rux of the mat ter

256 Cleveland Clinic Quar ter ly

of lipid accumulat ion in a therogene-sis. While the endothelial lining of an ar tery remains intact it func t ions as a remarkable protective ba r r i e r , not only excluding plasma l ipoproteins but also shielding the delicate meta-bolic balance of the tunica intima f r o m the pe r tu rb ing inf luences of o ther plasma prote ins . Once dam-aged or lost, it allows a series of events to take place involving se rum inf lux, increased lipid absorpt ion by intimal cells, esterification of choles-terol, blocking of d i f fus ion channels in the intercellular matr ix, degenera-tion of intimal cells, and the recruit-ment and prol i ferat ion of connective tissue cells in the int ima.

However , the regenerat ive capac-ity of endothe l ium is not insignifi-cant . A single t raumat ic event is un-likely on its own to result in a pe rma-nent loss of endothelial integri ty. Nei ther is it likely that the small inti-mal elevations which result f r o m a single in jury persist indefini tely. For the progressive degenera t ion and thickening of the intima which char-acterizes atherosclerosis, it makes sense to suggest that there must be a constant or at least repetit ive endo-thelial insult.

If such a sustained endothel ial in-ju ry is accepted as an inciter of ath-erosclerosis, it is clearly necessary to demons t ra te that such injuries can result f r o m the action of major risk factors . Fortunately, the question of whe ther hyper tens ion, c igarret te smoking, and hyper l ipopro te inemia can cause endothelial in jury is ame-nable to investigation. For example , recent studies have associated the a therogenic effects of tobacco smoke with its carbon monoxide content . 3 7

Exper iments in rabbits38 and pri-mates39 exposed to relatively small

Vol. 43, No. 3

concentrat ions of carbon monoxide (0.0009-0.025 vol %) for per iods of a few weeks have shown that the initial reaction is one of endothel ia l in jury . T h e endothelial cells swell and be-come disorganized, the in terendo-thelial gaps a p p e a r widened, and there is considerable edema of the subendothe l ium. T h e s e changes have also been seen in the umbilical veins of infants bo rn to smoking mothers but not in those of nonsmok-ing mothers . 4 0

Exper imental work has also unde r -lined the sensitivity of the endothe-lium to high blood flow rates, in-creased shear stress, and he ightened pulse pressure , 4 1 all components of the hypertensive situation. Endo-thelial cells have been shown to be-come disorganized and even detached if the wall shear stress is high.4 2 Pres-sure related stresses on the endo-thelial layer may lower the threshold to shear damage . If excess vasoactive agents circulate in the hypertensive state, it is possible that they contribute to endothelial damage .

T h e reaction of the endothe l ium to hyperl ipidemia is more content ious, because it is difficult to separate the direct effects on endothelial cells f r o m the secondary response of the intima to deposited lipid. However , high concentrat ions of low density l ipoprotein appea r to be toxic to arte-rial cells in culture,4 3 and they may well be so to endothelial cells in vivo. However , it is not necessary to postu-late direct endothel ial toxicity in hy-per l ipoprote inemia , for the height-ened blood viscosity occurr ing in this situation is likely to br ing about cell disrupt ion by causing increased d rag effects on the endo the l ium.

Finally, since the initial observa-tions by Goldenberg et al44 that endo-

Winter 1976 Revised concepts of atherogenesis 257

thelial proliferation is a characteristic fea ture of diabetic angiopathy, it has become increasingly clear that vascu-lar endothel ium is highly susceptible to damage in diabetes mellitus45 and that endothelial injury may be the initiating event in the widespread vascular complications of this dis-ease.4"'47 It remains unclear whether endothelial damage in diabetes is sec-ondary to abnormally high levels of circulating metabolites such as glu-cose or lactate, or whether the re is an integral alteration in cell metabolism resulting in a lowered resistance to injury and premature senescence. Whichever is the case, such effects may also be present in persons who have varying degrees of glucose intol-erance without overt diabetes, thus accounting for the significance of this metabolic abnormality in the epide-miology of atherosclerotic disease.

Endothelial injury the re fore ap-pears to represent a common path-way by which each of the major risk factors might contribute to the initia-tion of atherosclerosis. This view is suppor ted by a number of recent in-vestigations in which changes in inti-mal morphology have been studied af ter artificial endothelial in jury. Al-though atherogenesis is undoubtedly a complicated process in which many aspects of intimal morphology and metabolism are disturbed, loss of en-dothelial integrity seems pa ramoun t . I suggest that in many respects ath-erosclerosis may be regarded as a chronic endotheliopathy.

Proliferation and fibrosis within the intima

If a careful microscopic examina-tion is made of the diseased segments of the coronary artery that are re-moved surgically f rom patients un-

dergoing coronary reconstructive surgery, or of those that are dissected postmortem f r o m patients who have died f rom occlusive coronary disease, it will be seen that the bulk of the occluding lesion is usually composed of either dense fibrous connective tis-sue similar to that shown in Figures 1 and 2 or of thrombotic material simi-lar to that blocking the lumen in Fig-ure 5. Even when sudden thrombosis occurs, it invariably does so in an ar-tery that is already narrowed by fi-brous connective tissue (Fig. 5). Thus , a large par t of the clinical syn-d r o m e of reduced coronary blood flow appears to be a result of chronic stenosing fibrous plaques within the coronary arterial tree.48 This is not to say that the lesions show no lipid ac-cumulation, nor that thrombosis may not produce the precipitating crisis, but ra ther that we have tended to disregard the fibrosis which is so characteristic of these lesions.

What causes such p ro found intimal fibrosis? T h e r e are two schools of thought . One is that the presence of cholesterol, and certain saturated lip-ids stimulate the proliferation of smooth muscle cells in the intima and that these cells are "modif ied" in the process into active connective tissue synthesizing cells. T h e other school of thought is based on the pro-nounced tendency of h u m a n athero-sclerotic lesions to r u p t u r e and split, producing areas of in t ramural hem-orrhage , mural thrombosis necrosis and calcification.49 T h e fibrosis which ensues can be regarded as a repair reaction to r u p t u r e and necrosis, or as a foreign body reaction, or as an organization of mural hemor rhage and thrombosis. This second view has received less suppor t f rom the atherosclerosis community than

258 Cleveland Clinic Quar ter ly Vol. 43, No. 3

F i g . 5. H u m a n c o r o n a r y a r t e r y . R e c e n t t h r o m b o t i c o c c l u s i o n o f a n a r t e r y a l r e a d y s t e n o s e d by a d v a n c e d f i b r o u s a t h e r o s c l e r o s i s ( h e m a t o x y l i n a n d e o s i n s t a i n , X40) .

would be expected f r o m an examina-tion of human lesions, partly because h e m o r r h a g e within plaques is un-common in exper imenta l animal le-sions.

I see no reason to separate these events. Fibrosis is characterized in all locations by a dramat ic accumulat ion of f ibrous collagen in the intercellu-lar matr ix. T h e critical question is what is the mechanism which controls collagen product ion and deposition in pathologic sites and how is it acti-vated by these d i f f e ren t stimuli.

Recent studies with aortic smooth muscle cells in cul ture suggest that l ipoproteins, isolated f r o m hyperli-popro te inemic se rum, can directly stimulate cell proliferation.5 0 , 5 1 How-ever, increased numbers of smooth muscle cells do not inevitably imply increased collagen biosynthesis. So far , se rum l ipoproteins have not been

shown to initiate increases in arterial collagen synthesis,52 a l though dra-matic elevations in the rate of colla-gen fo rmat ion have been observed in exper imenta l atherosclerotic lesions p roduced u n d e r hyper l ipoprote i -nemic conditions.5 3 - 5 5 T h e Table sum-marizes much of the evidence for en-hanced collagen synthesis in a thero-sclerotic lesions and compares rates of synthesis and levels of enzyme stimulation within diseased arterial segments with values obtained f r o m adjacent normal segments . Theore t i -cally, it seems reasonable to propose that activators of fibrosis exist within atherosclerotic plaques, a l though their na tu re and origin a re presently obscure. Some investigators56 suggest that the enzyme prolyl hydroxylase is an impor tan t regulatory step in colla-gen biosynthesis. Increased prolyl hydroxylase activity has been demon-

Winter 1976 Revised concepts of atherogenesis 259

Spccies

T a b l e . I n c r e a s e s in a o r t i c c o l l a g e n s y n t h e s i s in e x p e r i m e n t a l a t h e r o s c l e r o s i s

Elevation in ra te of collagen synthesis.

Ratio of athero-sclerotic tissue to

normal Atherogenic regimen Assay procedure

1 4 C - h y p r o 1 4 C - h y p r o P H act ivi ty P H act ivi ty P H activity P H act ivi ty 1 4 C - h y p r o P H activity

D o g 53

Rabb i t 5 4

R a b b i t " R a b b i t 5 8

Pig 5 9

Pig 5 9

P i g e o n 5 5

P i g e o n 6 0

H C d i e t H C d i e t E p i n e p h r i n e t h y r o x i n e M e c h a n i c a l i n j u r y H C d i e t N o r e p i n e p h r i n e t h y r o x i n e H C d i e t H C d i e t

5 .2 1.7 5 .2 2.6

3.0 2.8

3 .3 1.0

H C d i e t = h y p e r c h o l e s t e r o l e m i c d i e t ; 1 4 C - h y p r o = c o l l a g e n syn thes i s a s s a y e d by m e a s u r i n g t h e i n c o r p o r a t i o n of 1 4 C - p r o l i n e i n t o p e p t i d e b o u n d 1 4 C - h y d r o x y p r o l i n e ; P H act ivi ty = p ro ly l h y d r o x y l -ase act ivi ty a s sayed in t h e t i s sue . R a t e s of c o l l a g e n s y n t h e s i s a r e a s s u m e d t o p a r a l l e l c h a n g e s in t h e activity of th i s e n z y m e in t i s sues .

strated in several d i f fe ren t models of experimental atherosclerosis.57-69

T h e cholesterol and lard fed pigeon appears at present to be anomalous6 0

{Table). Prolyl hydroxylase is similarly stimulated in fibrosing liver, and chemical activators of the enzyme have been isolated f r o m this tissue,61

but no such activator has yet been identified in atherosclerotic tissues.

Progression and regression of athero-sclerotic lesions

Stemerman and Ross62 showed re-cently in an interesting exper iment with macaques, that if the abdominal aorta and iliac arteries are denuded of their endothel ium with an intraar-terial catheter, there is immediate in-flux of plasma proteins, degenera-tion of subendothelial intimal cells, and migration of smooth muscle cells f rom the media into the damaged in-tima. T h e result is the formation of a fibroproliferative thickening of the intima. It is interesting that af ter about 6 months these intimal thicken-ings are no longer visible. T h e intima reverts to its normal width of a few cells, the endothel ium is regener-ated, and there is apparent ly little

indication of the previous injury.6 3

However, if a sustained hypercholes-terolemia is produced by feeding the animals a high fat diet t h roughou t the exper iment , the lesions do not regress but develop into lipid-rich fi-brous plaques similar to those seen in man.6 3

Two conclusions can be drawn f r o m these experiments . First, that even with an exceptionally severe sin-gle intimal injury, reparative intimal thickening is followed by a phase of regression and regenerat ion; and second, that lipid inf lux is a potent stimulus to the development of fi-brous plaques. T h e importance of hyperl ipoproteinemia has already been discussed, but the capacity of the arterial wall for regenerat ion is another mat ter . It suggests that le-sions produced unde r natural condi-tions may also be susceptible to modi-fication when the causative influ-ences are removed. It implies a cer-tain amount of optimism in the mod-erat ing effects of reducing risk factor levels in individuals and in the popu-lation as a whole.

This suggests that the natural his-tory of an atherosclerotic lesion is one

260 Cleveland Clinic Quarterly Vol. 43, No. 3

of proliferative response followed by stabilization and eventual remodel-ing in much the same way as a bone f rac ture heals by proliferative union, regenerat ion, and remodel ing. This is t rue of minor intimal lesions such as fatty streaks. Mitchell and Schwartz64 clearly documented this some years ago by demonstra t ing that a l though the number of fatty streaks in the human aorta may in-crease with age, their distribution may change. Fur the rmore , fibrous plaques do not necessarily develop in the sites where fatty streaking is most common at an early age. One must assume that many of the original fatty streaks regress spontaneously. It is more controversial to adopt a similar attitude to advanced fibro-fatty lesions, but in other n o n h u m a n primate experiments,6 5 occlusive ath-eromatous lesions produced by feed-ing a high-fat diet for prolonged pe-riods have been induced to regress to much smaller fibrous thickenings by a relatively short postexperimental period of low-fat feeding. It is possi-ble that lengthening the regression period would bring about a compara-ble f u r t h e r reduct ion of the f ibrous scar.

T o some extent this a rgumen t is academic in that it is much more dif-ficult to reduce effectively the levels of hyperl ipoproteinemia, hyper ten-sion, and cigarette smoking in the human populat ion than it is in the experimental monkey. One of the reasons for the steady progression of human atherosclerosis with age is that the inciting stimuli of ten remain active th roughou t , and the lesions are therefore never given the chance to remodel . What I have talked about as the natural history of the athero-sclerotic plaque may be prevented

f rom occurring unde r the high risk conditions of western society. Whether we can restore the natural pat tern of regression by therapeut ic means remains to be seen f r o m the results of primary prevention trials.

Conclusion —a unified theory of premature atherosclerosis

If what has gone before is ac-cepted, it is evident that there is no inherent biological reason why ath-erosclerosis should be a progressive disease. Arteries are capable of un-dergoing healing following an injury in a fashion similar to other tissues. Neither is there any immutable rea-son why occlusive arterial disease should be responsible for such a ma-jo r share of the causes of human death in developed countries. It is not so in the Masai66 and neither is it so in elephants.6 7 Disease surveys in the latter species have shown that the development of a limited amoun t of f ibrous atherosclerosis may be inevi-table dur ing a prolonged life. It is equally clear that the uncomplicated spontaneous atherosclerosis that oc-curs in this and other species is not of ten a cause of death in animals. Such lesions as occur spontaneously in animals have been re fe r red to as prototypes of the uncomplicated hu-man lesion.68 With this in mind, the complex problems of h u m a n athero-genesis can be distilled into one ra ther demanding question. What is it which in many middle-aged people living in industrial societies converts a relatively harmless prototype lesion into a complicated occlusive "malig-nant" process which destroys the nor-mal arterial s tructure and brings about p remature death? Despite re-cent evidence of a monoclonal origin of atherosclerotic plaques89 viral

Winter 1976

t ransformat ion seems unlikely. At our present state of knowledge,

the most sensible answer to this ques-tion would appea r to be a cont inued or of ten repea ted abuse or insult to arterial endo the l ium. T h e major risk factors: hyper l ip idemia, hyper ten-sion, glucose intolerance, and ciga-ret te smoking must play a major par t in this abuse. Each in its own way brings about and sustains endothelial in jury , and this is probably of major impor tance in de te rmin ing the fate of initially minor and uncomplicated lesions. Figure 6 emphasizes and sum-marizes diagrammatically this con-cept of atherogenesis . T h e u p p e r cy-cle of fou r pictures (A-D) represents the development of focal or d i f fuse intimal plaques or thickenings within coronary arteries in response to re-petetive endothel iopathy. It is proba-bly rational to suggest as Stehbens7 0

has done that most initial injur ies arc hemodynamic , arising f r o m normal "wear and tear" on the arterial wall, a l though there is no reason why pri-mary lesions might not originate f r o m toxic injuries to the endothe-lium or p r ema tu re endothelial senes-cence.

Initial damage to the endothe l ium allows an influx of plasma lipopro-teins, a lbumin, f ibr inogen and other macromolecules, many of which be-come enmeshed in the hyaluronate gel of the intimal g r o u n d substance. T h e alteration of the microenviron-ment will directly affect many intimal cells, while the large n u m b e r s of se-questered plasma macromolecules will in te r fe re with the d i f fus ion of oxygen and nutr ients t h r o u g h the tis-sue. This inevitably results in cellular degenera t ion in the int ima and the liberation of in f lammatory sub-stances, metabolic activators, and mi-

Revised concepts of a therogenesis 261

togens. T h e metabolic and prol ifera-tive reaction to these events is de-picted in d iagram 6C. T h e plaque is f o r m e d f r o m prol i ferat ion of acti-vated intimal cells, possible recruit-men t and t rans format ion of smooth muscle cells f r o m the media , and en-hanced synthesis and accumulat ion of intimal connective tissue. Lipid within the lesion is derived mainly f r o m sequestered l ipoproteins , but there is also an activation of several aspects of lipid metabolism in the newly prol i fera t ing intimal cells.

T h e endothelial reaction is likely to d e p e n d on the degree of in jury: se-verely d a m a g e d cells will lose their a t tachment to the basement mem-b r a n e and a re lost; less d a m a g e d cells n o doubt recover . Platelet thrombi f o r m at areas of endothel ial denuda -tion a n d endothel ial regenera t ion en-sues. Failing f u r t h e r in jury , the en-dothelial barr ier is reestablished and ent ry of plasma macromolecules halts. F rom this point on , a phase of healing occurs which is characterized not only by endothelial integrity but also by reorganizat ion of the reac-tively thickened int ima. With time there is a migration of cells away f r o m the lesion and partial catabo-lism of the newly synthesized connec-tive tissue. Regression is unlikely to be complete , and yet connective tis-sue remode l ing may occur to a grea ter extent than exper imenta l work has yet indicated.

T h e critical stage in the a theroge-netic process is the impa i rmen t of this no rmal process of healing and regenera t ion and its rep lacement by a cont inuous cycle of cell damage and metabolic st imulation. Figure 6E shows how sustained hyper l ip idemia, hyper tens ion , carboxyhemoglobine-mia, or any o ther cause of cont inuous

262 Cleveland Clinic Quar ter ly

A. N O R M A L ARTERY

Vol. 43, No. 4

D. HEALING A N D REGRESSION

Endothelial injury \ High shear stress

carbon monoxide circulating amines hyperlipemia?

No further injury

B. ENDOTHELIAL DISRUPTION

• S i ; ¿ T •

C. REACTIVE PHASE

Persistent hypertension Hyperlipemia or

cigarette smoking

E. ACTIVE ATHEROSCLEROSIS

x ç i W /

F i g . 6 . A r t e r i a l r e a c t i o n s a n d a t h e r o g e n e s i s . A - D s tages in t h e n o r m a l cycle o f i n t i m a i i n j u r y a n d

A P l i r p i a s m a m a c r o m o l e c u l e s ; 2. i n t ac t c o n t i n u o u s e n d o t h e l i u m ; 3 . n o r m a l h u m a n ^ i n t i m a c o n t a i n -i n g fibroblasts, s m o o t h m u s c l e cells , m a c r o p h a g e s a n d f i n e c o n n e c t é e Ussue s t r o m a , 4 . f e n e s -

Winter 1976 Revised concepts of a therogenesis 263

in jury is likely to p revent endothel ial recovery and regenera t ion and en-courage m o r e p r o f o u n d mura l thrombosis . Cont inued inf lux of plasma maintains the st imulus for plaque en la rgement . Rapid impair-men t of d i f fus ion within the plaque results in cell necrosis. Lipoprotein degenera t ion creates an abundance of insoluble lipid. Reactive fibrosis continues at a high ra te , and the ex-pand ing bulk of the lesion en-croaches on the arterial l u m e n .

This is admittedly a simplistic view of the a therogenic process and yet it represents a coheren t theory . Each risk factor is viewed as contr ibut ing to endothelial in jury , and p laque de-velopment is seen as primari ly a reac-tion to changes in the int ima b rough t about by endothelial damage . This theory has the advantage of not plac-ing great emphasis on mult iple path-ways in the pathogenesis of the dis-ease. T h e search for one basic path-

way of atherogenesis may well be elu-sive, yet it is valuable for its d e m a n d s that apparen t ly opposing results be reconciled.

If the theory of atherosclerosis as a chronic arterial endothe l iopa thy is correct , some opt imism may be per-mitted in viewing the potential devel-o p m e n t of control measures to pre-vent the disease. All of the presently r e c o m m e n d e d intervent ions fo r low-er ing major risk factors in individuals and in populat ions can be expected to r educe the incidence of endo the -lial in jury and at least slow the rate of plaque deve lopment . A similar but more direct approach to prevent ion might be to search for agents which will protect the endo the l ium against low grade in jury . Failing this, it might be feasible to diminish the re-sponse of subendothelial int imal tis-sue to endothel ial damage . This might be achieved by inhibit ing plate-let func t ion with d rugs such as dipyr-

t r a t e d i n t e r n a l e last ic l a m i n a ; 5. m e d i a l s m o o t h m u s c l e , e las t in a n d c o l l a g e n a r r a n g e d in c o n c e n t r i c l a y e r s .

B . 1. P l a s m a m a c r o m o l e c u l e s ; 2. loss of e n d o t h e l i a l i n t e g r i t y —cells swo l l en a n d i n t e r c e l l u l a r j u n c -t ions d i s r u p t e d ; 3 . i n f l u x o f p l a s m a m a c r o m o l e c u l e s i n t o c o n n e c t i v e t i s sue of i n t i m a ; 4 . f e n e s -t r a t e d i n t e r n a l e las t ic l a m i n a ; 5. s o m e p l a s m a c o n s t i t u e n t s r e a c h m e d i a , o t h e r s r e m a i n e n m e s h e d in i n t i m a .

C . 1. P l a s m a m a c r o m o l e c u l e s ; 2 . p l a t e l e t t h r o m b i f o r m at s i tes o f i n j u r y ; s eve re ly d a m a g e d e n d o t h e -lial cells a r e los t , r e g e n e r a t i v e c h a n g e s o c c u r in o t h e r s ; 3. r e ac t i ve c h a n g e s o c c u r in i n t i m a : cells p r o l i f e r a t e a n d / o r b e c o m e me tabo l i ca l l y m o r e ac t ive , s m o o t h m u s c l e cells m i g r a t e i n t o i n t i m a f r o m m e d i a a n d c h a n g e t o r e s e m b l e f i b r o b l a s t s ; m a c r o p h a g e s e n g u l f t r a p p e d m a c r o m o l e c u l e s , a n d t h e r e is h e i g h t e n e d s y n t h e s i s o f c o n n e c t i v e t i s sue in t h e i n t i m a ; 4. i n t e r n a l e las t ic l a m i n a r e m a i n s i n t ac t ; 5. p l a s m a c o n s t i t u e n t s in m e d i a d i f f u s e i n t o l y m p h a t i c s .

D. 1. P l a s m a m a c r o m o l e c u l e s ; 2 . e n d o t h e l i a l i n t e g r i t y r e s t o r e d ; 3. m a j o r i t y of p l a s m a c o m p o n e n t s r e m o v e d f r o m i n t i m a , s o m e low d e n s i t y l i p o p r o t e i n a n d f i b r i n o g e n r e m a i n ; m a n y of t h e m o d i f i e d s m o o t h m u s c l e cells m i g r a t e f r o m t h i c k e n e d i n t i m a , a n d m u c h of excess i n t i m a l c o n n e c t i v e t i s sue is c a t a b o l i z e d ; 4. i n t e r n a l e las t ic l a m i n a ; 5. n o r e s i d u a l c h a n g e s in t u n i c a m e d i a .

E . P r o g r e s s i v e a t h e r o s c l e r o t i c l e s ion p r o d u c e d by c o n t i n u e d i n t i m a l i n j u r y . 1. P l a s m a m a c r o m o l e -cules ; 2. c o n t i n u e d i n j u r y p r e v e n t s e n d o t h e l i a l r e g e n e r a t i o n a n d i n c r e a s e d p e r m e a b i l i t y pe r s i s t s ; 3. m u r a l t h r o m b i f o r m w h e r e s u b e n d o t h e l i a l t i s sue c o n t i n u e s to be e x p o s e d ; 4 . " f i b r o b l a s t - I i k e " cells in i n t i m a a r e a c t i v a t e d t o s e c r e t e l a r g e a m o u n t s of c o n n e c t i v e t i s sue ; 5 . f o a m cells f o r m a n d l ip id m e t a b o l i s m is d e r a n g e d in i n t i m a l cells; 6 . c o n n e c t i v e t i s sue of i n t i m a b e c o m e s p r e d o m i -n a n t l y c o l l a g e n o u s ; 7. a r e a s of h e m o r r h a g e w i t h i n t h e p l a q u e o c c u r ; 8. c h o l e s t e r o l c rys ta l s p r e c i p i t a t e in p l a q u e a n d f o r m " c l e f t s " in c o n n e c t i v e t i s sue ; 9 . l ip id col lects a t b a s e of les ion in a m o r p h o u s " p o o l s " ; 10. n e c r o s i s o f i n t i m a l cells a n d d e g e n e r a t i o n of i n t e r n a l e las t ic l a m i n a ; 11. a n o x i c d e g e n e r a t i o n o c c u r s in s o m e m e d i a l cells b e n e a t h t h e p l a q u e .

264 Cleveland Clinic Quarterly

idamole or aspirin, as suggested by Spaet,71 or by inhibiting the prolifer-ative and synthetic responses within the intima with agents which are spe-cific and nontoxic. Urgent priority should be given to the development of such agents and to basic research which will f u r t he r knowledge of the cellular and molecular causes of the disease. If the results of such re-search are directed towards a contin-ued reassessment of cur ren t theories of atherogenesis, there is every rea-son to believe that funct ional aspects of plaque development will soon be unders tood and effective control of atherosclerotic disease brought nearer .

References 1. P a g e I H : A t h e r o s c l e r o s i s : a n i n t r o d u c t i o n .

C i r c u l a t i o n 10: 1 - 2 7 , 1954. 2 . P a g e I H : A r t e r i a l h y p e r t e n s i o n in r e t r o -

s p e c t . C i r c Res 34: 133-142 , 1974. 3. K o t i n P: C a n c e r : a d i s e a s e i n v o l v i n g mu l t i -

p l e f a c t o r s , in M u l t i p l e F a c t o r s in t h e C a u -s a t i o n of E n v i r o n m e n t a l l y I n d u c e d Dis-e a s e . L e e K H K , K o t i n P , e d s . N e w Y o r k , A c a d e m i c P r e s s , 1972, p p 15-27 .

4 . S i m b o r g D W : T h e s t a t u s of r i sk f a c t o r s a n d c o r o n a r y h e a r t d i s e a s e . J C h r o n i c Dis 22: 5 1 5 - 5 5 2 , 1970.

5. K a n n e l W B , G o r d o n T : T h e F r a m i n g h a m S t u d y . A n E p i d e m i o l o g i c a l I n v e s t i g a t i o n of C a r d i o v a s c u l a r D i sease , S e c t i o n s 1 - 2 7 , W a s h i n g t o n , U . S . G o v e r n m e n t P r i n t i n g O f f i c e , 1971.

6 . I n t e r - S o c i e t y C o m m i s s i o n f o r H e a r t Dis-e a s e R e s o u r c e s , A t h e r o s c l e r o s i s S t u d y G r o u p a n d E p i d e m i o l o g y S t u d y G r o u p : P r i m a r y p r e v e n t i o n o f t h e a t h e r o s c l e r o t i c d i s ea se s . C i r c u l a t i o n 42: A 5 5 - 9 5 , 1970.

7. N a t i o n a l H e a r t a n d L u n g I n s t i t u t e T a s k F o r c e o n A r t e r i o s c l e r o s i s : A r t e r i o s c l e r o s i s , Vol I I , W a s h i n g t o n , D e p a r t m e n t of H e a l t h , E d u c a t i o n a n d W e l f a r e , 1971.

8. Keys A: C o r o n a r y h e a r t d i s e a s e in s e v e n c o u n t r i e s . C i r c u l a t i o n 41: S u p p l I : 1 - 2 1 1 , 1970.

9 . S m i t h E B , Evans P H , D o w n h a m M D : L i p i d in t h e ao r t i c i n t i m a ; t h e c o r r e l a t i o n o f m o r p h o l o g i c a l a n d c h e m i c a l c h a r a c t e r -

Vol. 43, No. 3

is t ics. J A t h e r o s c l Res 7: 171 -186 , 1967. 10. S m i t h E B , S l a t e r R S , C h u P K : T h e l i p i d s

in r a i s e d f a t t y a n d f i b r o u s l e s i o n s in h u -m a n a o r t a . A c o m p a r i s o n of t h e c h a n g e s a t d i f f e r e n t s t a g e s of d e v e l o p m e n t . J A t h e r -oscl Res 8: 399-419 , 1968.

11. S m i t h E B , S la te r RS: T h e m i c r o d i s s e c t i o n of l a r g e a t h e r o s c l e r o t i c p l a q u e s t o give m o r p h o l o g i c a l a n d t o p o g r a p h i c a l l y d e -f i n e d f r a c t i o n s f o r ana lys i s . P a r t I . T h e l ip ids in t h e i so la t ed f r a c t i o n s . A t h e r o s c l e -ros is 15: 3 7 - 5 6 , 1972.

12. S m i t h E B : T h e r e l a t i o n s h i p b e t w e e n p l a s m a a n d t i s sue l ip ids in h u m a n a t h e r o -sc le ros i s . A d v L ip id Res 12: 1 - 4 9 , 1974.

13. B u t k u s A , L a z z a r i n i R o b e r t s o n A , E h r h a r t L A , et al: A o r t i c l ip ids a t d i f f e r e n t s t ages o f c a n i n e e x p e r i m e n t a l a r t e r i o -sc le ros i s . E x p Mol P a t h o l 16: 3 1 1 - 3 2 5 , 1972.

14. D a y t o n S, H a s h i m o t o S: C h o l e s t e r o l flux a n d m e t a b o l i s m in a r t e r i a l t i s sue a n d in a t h e r o m a t a . E x p Mol P a t h o l 13: 2 5 3 - 2 6 8 , 1970.

15. C h o b a n i a n A V : S t e r o l s y n t h e s i s in t h e h u -m a n a r t e r i a l i n t i m a . J Clin I n v e s t 47: 595 -603, 1968.

16. F ie ld H , Swell L , S c h o o l s P E J r , e t al: D y n a m i c a s p e c t s of c h o l e s t e r o l m e t a b o l i s m in d i f f e r e n t a r e a s o f t h e a o r t a a n d o t h e r t i s sues in m a n a n d t h e i r r e l a t i o n s h i p t o a t h e r o s c l e r o s i s . C i r c u l a t i o n 22: 5 4 7 - 5 5 8 , 1960.

17. Wa t t s H F : Ro le o f l i p o p r o t e i n s in t h e f o r -m a t i o n o f a t h e r o s c l e r o t i c l e s i o n s , in E v o l u -t ion o f t h e A t h e r o s c l e r o t i c P l a q u e . J o n e s R J , e d . C h i c a g o , U n i v e r s i t y o f C h i c a g o P r e s s , 1963, p p 117-126.

18. K a o V C Y , Wiss le r R W : A s t u d y o f t h e i m m u n o h i s t o c h e m i c a l l oca l i za t ion of se-r u m l i p o p r o t e i n s a n d o t h e r p l a s m a p r o -t e ins in h u m a n a t h e r o s c l e r o t i c l e s i o n s . E x p Mol P a t h o l 4: 4 6 5 - 4 7 9 , 1965.

19. W o o l f N , P i l k i n g t o n T R E : T h e i m m u n o -h i s t o c h e m i c a l d e m o n s t r a t i o n of l i p o p r o -te ins in vessel walls . J P a t h Bac t 90: 4 5 9 -463, 1965.

20 . W a l t o n K W , W i l l i a m s o n N : His to log ica l a n d i m m u n o f l u o r e s c e n t s t u d i e s o n t h e ev-o l u t i o n of t h e h u m a n a t h e r o s c l e r o t i c p l a q u e . J A t h e r o s c l Res 8 : 5 9 9 - 6 2 4 , 1968.

21 . H o f f H F , H e i d e m a n C L , J a c k s o n R L , et al: Loca l i za t ion p a t t e r n s o f p l a s m a apo l i -p o p r o t e i n s in h u m a n a t h e r o s c l e r o t i c le-s ions . C i r c u l a t i o n Res 37: 7 2 - 7 9 , 1975.

22 . G e r o S, G e r g e l y J , J a k a b L , e t al: C o m p a r -

Winter 1976 Revised concepts of a therogenesis 265

ative i m m u n o e l e c t r o p h o r e t i c s tud ies o n h o m o g e n a t e s o f a o r t a , p u l m o n a r y a r t e r i e s a n d i n f e r i o r v e n a cava of a t h e r o s c l e r o t i c i n d i v i d u a l s . J A t h e r o s c l Res 1: 88 -91 , 1961,

23. Smi th E B , S la te r R: T h e c h e m i c a l a n d im-m u n o l o g i c a l assay of low dens i ty l i p o p r o -teins e x t r a c t e d f r o m h u m a n ao r t i c i n t i m a . A the rosc l e ros i s 11: 417-428 , 1970.

24. Smi th E B , A l e x a n d e r K M , Mass ie IB: Q u a n t i t a t i v e s t u d i e s o n t h e r e l a t i o n s h i p s b e t w e e n inso lub le ' f i b r i n ' , so lub le f i b r i n o -g e n a n d low d e n s i t y l i p o p r o t e i n . A t h e r o -sclerosis 23: 19-39, 1976.

25. F lorey H , S h e p p a r d B L : T h e p e r m e a b i l i t y of a r t e r i a l e n d o t h e l i u m to h o r s e r a d i s h p e r o x i d e . P roc R Soc L o n d (Biol) 174: 435 -443, 1970.

26. Reese T S , K a r n o v s k y MJ: F ine s t r u c t u r a l local izat ion of a b l o o d b r a i n b a r r i e r to ex-o g e n o u s p e r o x i d a s e . J Cell Biol 34: 207 -217, 1967.

27. H u t t n e r I , B o u t e t M , M o r e R H : S tud ie s o n p r o t e i n p a s s a g e t h r o u g h a r t e r i a l e n d o -t h e l i u m . I . S t r u c t u r a l co r r e l a t e s of p e r m e -ability in ra t a r t e r i a l e n d o t h e l i u m . L a b In -vest 28: 672-677 , 1973.

28. Schwar t z SM, B e n d i t t EP : S tud ie s o n a o r -tic i n t i m a . I . S t r u c t u r e a n d p e r m e a b i l i t y of r a t t h o r a c i c aor t i c i n t i m a . A m J Pa tho l 66: 241-264, 1972.

29. H u t t n e r I , B o u t e t M, R o n a G , e t al: S t u d -ies o n p r o t e i n p a s s a g e t h r o u g h a r t e r i a l e n d o t h e l i u m . I I I . E f f e c t o f b lood p r e s s u r e levels o n t h e pas sage of f i n e s t r u c t u r a l p r o t e i n t r a c e r s t h r o u g h r a t a r t e r i a l e n d o -t h e l i u m . L a b Inves t 29: 536-546 , 1973.

30. R o b e r t s o n A L , K h a i r a l l a h P A : Ar t e r i a l en -do the l i a l p e r m e a b i l i t y a n d vascu la r dis-ease . T h e t r a p d o o r e f f e c t . E x p Mol P a t h o l 18: 241-260 , 1973.

31. Cas ley-Smi th J R , C h i n J C : T h e pas sage of cy top la smic vesicles across e n d o t h e l i a l a n d meso the l i a l cells. J Microsc ( O x f ) 93: 169-189, 1971.

32. S c a n u A M ; T h e s t r u c t u r e o f h u m a n s e r u m low a n d h i g h dens i ty l i p o p r o t e i n s , in A t h -e r o g e n e s i s : I n i t i a t i n g Fac to r s , Ciba F o u n -d a t i o n S y m p o s i u m 12 (new ser ies ) , A m -s t e r d a m , Assoc ia ted Scient i f ic Pub l i she r s , 1973, p p 223-246 .

33. B u c k RC: T h e f i n e s t r u c t u r e o f t h e aor t ic e n d o t h e l i a l les ions in e x p e r i m e n t a l choles-te ro l a the rosc le ros i s of r abb i t s . A m J Pa-thol 34: 897-909 , 1958.

34. P a r k e r F, O d l a n d G F : A cor re l a t ive his to-c h e m i c a l , b i o c h e m i c a l a n d e l e c t r o n micro-

scopic s t u d y o f e x p e r i m e n t a l a t h e r o s c l e r o -sis in t h e r abb i t a o r t a with specia l r e f e r -e n c e to t h e m y o i n t i m a l cell. A m J P a t h o l 48: 197-239, 1966.

35. S i lkwor th J B , M c L e a n B, S t e h b e n s WE: T h e e f f e c t of h y p e r c h o l e s t e r o l e m i a o n aor t i c e n d o t h e l i u m s t u d i e d en face. A th -erosc leros is 22: 335-348, 1975.

36. W e b e r G, Fab r in i P , Capacc io l i E , e t al: R e p a i r of ear ly c h o l e s t e r o l - i n d u c e d ao r t i c les ions in rabbi t s a f t e r w i t h d r a w a l f r o m s h o r t t e r m a t h e r o g e n i c d i e t . A t h e r o s c l e r o -sis 22: 565-572, 1975.

37. A s t r u p P, K j e l d s e n K: C a r b o n m o n o x i d e , s m o k i n g a n d a t h e r o s c l e r o s i s . S y m p o s i u m o n A the rosc l e ro s i s . M e d Clin N o r t h A m 58: 323-350, 1974.

38. K j e l d s e n K, A s t r u p P, W a n s t r u p J : U l t r a -s t r u c t u r a l i n t i m a l c h a n g e s in t h e r a b b i t a o r t a a f t e r a m o d e r a t e c a r b o n m o n o x i d e e x p o s u r e . A the rosc l e ros i s 16: 6 7 - 8 2 , 1972.

39 . T h o m s e n , K H : U l t r a s t r u c t u r a l c h a n g e s in t h e c o r o n a r y a r t e r i e s o f p r i m a t e s (Macaca iris) a f t e r a p r o l o n g e d , mi ld c a r b o n m o n -o x i d e e x p o s u r e , in A the rosc l e ros i s I I I . Sche t t l e r G, e d . A Weizel , B e r l i n , S p r i n -g e r - V e r l a g , 1974, p p 333-335 .

40. A s m u s s e n I , K je ld sen K: I n t i m a l u l t r a -s t r u c t u r e of h u m a n umbi l ica l a r t e r i e s ; ob-se rva t i ons o n a r t e r i e s f r o m n e w b o r n chil-d r e n of s m o k i n g a n d u n s m o k i n g m o t h e r s . C i rc Res 36: 579-589, 1975.

41 . Fry DL: R e s p o n s e s of t h e a r t e r i a l wall to ce r t a in physical f ac to r s , in A t h e r o g e n e s i s : I n i t i a t i ng Fac to r s , Ciba F o u n d a t i o n Sym-p o s i u m 12 (new ser ies) , A m s t e r d a m , Asso-c ia ted Scient i f ic P u b l i s h e r s , 1973, p p 93-125.

42 . Fry DL: A c u t e vascu la r e n d o t h e l i a l c h a n g e s associa ted with i n c r e a s e d b lood velocity g r a d i e n t s . Circ Res 22: 165-197, 1968.

43. L a z z a r i n i - R o b e r t s o n A: O x y g e n r e q u i r e -m e n t s of t h e h u m a n a r t e r i a l i n t i m a in a t h -e r o g e n e s i s . P r o g r B i o c h e m P h a r m a c o l 4: 3 0 5 - 3 1 9 , 1 9 6 8 .

44. G o l d e n b e r g S, Alex M, J o s h i R A , e t al: N o n - a t h e r o m a t o u s p e r i p h e r a l vascu la r d i sease of t h e lower e x t r e m i t y in d i abe t e s mel l i tus . Diabetes 8: 261-273 , 1959.

45. M o o r e J M , F rew I D O : P e r i p h e r a l vascu la r les ion in d iabe tes mel l i tus . Br M e d J 2: 19-23, 1965.

46 . V r a c k o R , B e n d i t t EP: Cap i l l a ry basal l am-ina t h i c k e n i n g : its r e l a t i o n s h i p to e n d o t h e -lial cell d e a t h a n d r e p l a c e m e n t . J Cell Biol

266 Cleveland Clinic Quar ter ly Vol. 43, No . 3

47: 2 8 1 - 2 8 5 , 1970. 47. V r a c k o R , B e n d i t t E P : M a n i f e s t a t i o n s of

d i a b e t e s me l l i t u s — t h e i r pos s ib l e r e l a t i o n -s h i p s t o an u n d e r l y i n g cell d e f e c t . A m J P a t h o l 75: 2 0 4 - 2 2 4 , 1974.

48. R o b e r t s W C : C o r o n a r y t h r o m b o s i s a n d fa-tal m y o c a r d i a l i s c h e m i a . C i r c u l a t i o n 49: 1 -3, 1974.

49 . F r i e d m a n M: P a t h o g e n e s i s o f C o r o n a r y A r t e r y D i sea se . N e w Y o r k , M c G r a w - H i l l , 1969.

50. D a o u d A S , Fr i t z K E , J a r m o l y c h T , e t al: U s e o f a o r t i c m e d i a l e x p l a n t s in t h e s t u d y o f a t h e r o s c l e r o s i s . E x p M o l P a t h o l 18: 177, 1973.

51. Ross R , G l o m s e t J A : A t h e r o s c l e r o s i s a n d t h e a r t e r i a l s m o o t h m u s c l e cel l . Sc i ence 180: 1332-1339 , 1973.

52. H o l d e r b a u m D , E h r h a r t L A , M c C u l l a g h K G : T h e e f f e c t s o f c h o l e s t e r o l - r i c h s e r u m l i p o p r o t e i n s a n d e x o g e n o u s p r o l i n e c o n -c e n t r a t i o n o n c o l l a g e n s y n t h e s i s by i s o l a t e d a o r t a s . P r o c Soc E x p Biol M e d 150: 3 6 3 -367, 1976.

53. M c C u l l a g h K G , E h r h a r t L A : I n c r e a s e d a r -t e r i a l c o l l a g e n in e x p e r i m e n t a l c a n i n e a t h -e r o s c l e r o s i s . A t h e r o s c l e r o s i s 19: 13-28 , 1974.

54. M c C u l l a g h K G , P a g e I H : I n c r e a s e d col la-g e n s y n t h e s i s in e a r l y r a b b i t a t h e r o s c l e r o -sis a n d its i n h i b i t i o n by c i s - h y d r o x y p r o -l i n e , in A t h e r o s c l e r o s i s I I I . S c h e t t l e r G , e d . A W e i z e l , B e r l i n , S p r i n g e r - V e r l a g , 1974, p p 2 3 9 - 2 4 1 .

55. M c C u l l a g h K G : A c t i v a t i o n o f c o l l a g e n syn -thes i s in a t h e r o s c l e r o t i c r e g i o n s of p i g e o n a o r t a s . C i r c u l a t i o n 50: S u p p l I I I : 268 , 1974.

56. G r a n t M E , P r o c k o p D J : T h e b i o s y n t h e s i s o f c o l l a g e n , p a r t I I I . N E n g l J M e d 286: 2 9 1 - 3 0 0 , 1972.

57. F u l l e r G C , L a n g n e r R O : E l e v a t i o n of a o r -tic p r o l i n e h y d r o x y l a s e : a b i o c h e m i c a l d e -f e c t in e x p e r i m e n t a l a r t e r i o s c l e r o s i s . Sci-e n c e 168: 9 8 7 - 9 8 9 , 1970.

58. L i n d y S, T u r t o H , U i t t o J , e t al: I n j u r y a n d r e p a i r in a r t e r i a l t i s sue in t h e r a b b i t . A n a l -ysis o f D N A , R N A , h y d r o x y p r o l i n e a n d l ac t a t e d e h y d r o g e n a s e in e x p e r i m e n t a l a r -t e r iosc le ros i s . C i r c Res 30: 123-130 , 1972.

59. Fu l l e r G C , Mil ler E , F a r b e r T , e t a l : A o r t i c c o n n e c t i v e t i s sue c h a n g e s in m i n i a t u r e

p igs f e d a l ipid r i ch d i e t . C o n n T i s s Res 1: 2 1 7 - 2 2 1 , 1972.

60 . St C la i r R W , T o m a J J , L o f l a n d H B : P r o -l i ne h y d r o x y l a s e activity a n d c o l l a g e n c o n -t e n t o f p i g e o n a o r t a s wi th n a t u r a l l y o c c u r -r i n g a n d c h o l e s t e r o l a g g r a v a t e d a t h e r o -sc le ros i s . A t h e r o s c l e r o s i s 21: 155-165 , 1975.

61 . M c G e e J O ' D , H a r e R P , P a t r i c k RS: S t i m u -l a t ion o f t h e c o l l a g e n b i o s y n t h e t i c p a t h w a y by f a c t o r s i so la t ed by e x p e r i m e n t a l l y in -

j u r e d r a t l i ve r . N a t u r e ( N e w Biol) 243: 121-123 , 1973.

62 . S t e m e r m a n M B , Ross R: E x p e r i m e n t a l a r -t e r i o s c l e r o s i s . I . F i b r o u s p l a q u e f o r m a t i o n in p r i m a t e s , a n e l e c t r o n m i c r o s c o p e s t u d y . J E x p M e d 136: 769-789 , 1972.

63 . Ross R , W i g h t T , G l o m s e t J : L e s i o n f o r -m a t i o n in h y p e r c h o l e s t e r e m i c p r i m a t e s f o l l o w i n g i n t r a v a s c u l a r e n d o t h e l i a l i z a t i o n , in A t h e r o s c l e r o s i s I I I . S c h e t t l e r G , e d . A W e i z e l , B e r l i n , S p r i n g e r - V e r l a g , 1974, p 860.

64. Mi tche l l J R A , S c h w a r t z CJ : A r t e r i a l Dis-e a s e , O x f o r d , Blackwel l , 1965.

65. A r m s t r o n g M L , W a r n e r E D , C o n n e r W E : R e g r e s s i o n o f c o r o n a r y a t h e r o m a t o s i s in r h e s u s m o n k e y s . Ci rc Res 27: 5 9 - 6 7 , 1970.

66. H o K J , B u s K, M i d d e l s o n B , e t al: T h e M a s a i o f Eas t A f r i c a : s o m e u n i q u e b io logi -cal c h a r a c t e r i s t i c s . A r c h P a t h o l 91: 3 8 7 -410, 1971.

67 . M c C u l l a g h K G : A r t e r i o s c l e r o s i s in t h e A f -r i c a n e l e p h a n t . P a r t I . I n t i m a l a t h e r o s c l e -rosis a n d its poss ib le c a u s e s . A t h e r o s c l e r o -sis 16: 307 -335 , 1972.

68 . G r e s h a m G A , H o w a r d A N : C o m p a r a t i v e h i s t o p a t h o l o g y o f t h e a t h e r o s c l e r o t i c le-s i o n . J A t h e r o s c l Res 3: 161-177 , 1963.

69. B e n d i t t EP : E v i d e n c e f o r a m o n o c l o n a l o r -ig in o f h u m a n a t h e r o s c l e r o t i c p l a q u e s a n d s o m e i m p l i c a t i o n s . C i r c u l a t i o n 50: 6 5 0 -652, 1974.

70. S t e h b e n s W E : H a e m o d y n a m i c p r o d u c t i o n o f l ip id d e p o s i t i o n , i n t i m a l t e a r s , m u r a l d i s sec t ion a n d t h r o m b o s i s in t h e b l o o d ves-sel wal l . P r o c R Soc L o n d (Biol) 185: 3 5 7 -373, 1974.

71. S p a e t T H : O p t i m i s m in t h e c o n t r o l o f a t h -e r o s c l e r o s i s . N E n g l J M e d 291: 5 7 6 - 5 7 7 , 1974.