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Localization in the human aortic wall of phospholipids containing choline
Journal of A therosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands L O C A L I Z A T I O N IN T H E HUMAN AORTIC W A L L OF P H O S P H O L I P I D S CONTAINING CHOLINE E. BOELSMA-VAN H O U T E AND C. J. F. BOTTCHER
Gaubius Institute of the University of Leyden, Leyden (The Netherlands) (Received September 26th, 1966)
SUMMARY
Qualitative histochemical investigations showed the following distribution of phospholipids containing choline in human aortas: (1) In the undiseased stage 0 aortas a positive reaction with cisaconitic anhydride was observed in: (a) smooth muscle cells of the intima and (b) smooth muscle cells and the elastic lamellae of the media. Positive reactions were sometimes seen in: (a) foam cells of the intima and (b) the internal elastic membrane. (2) In stage I aortas phospholipids containing choline were found in: (a) smooth muscle cells and foam cells in the intima and (b) foam cells in f a t t y streaks, (c) smooth muscle cells and elastic lamellae of the media. Occasional positive reactions were seen in: (a) the intercellular substance of the intima and (b) the internal elastic membrane. (3) In stage I I aortas phospholipids containing choline were found: (a) in foam cells of the f a t t y streaks, (b) in plaques intracellularly in the luminal margin, in myogenic foam cells (macrophages), and in connective tissue fibres, evenly spread in the undifferentiated mass of lipids with accumulations around cholesterol needles and deposits of calcium salts, in degenerated elastic lamellae, (c) in the media in smooth muscle cells and in the irregularly shaped nuclei as well as in the elastic lamellae. (4) The distribution in stage I I I aortas was identical to that in aortas of stage II; complicated lesions were not investigated. Some hypothetical statements about the origin and development of atherosclerotic lesions are discussed.
INTRODUCTION Previous papersl, 2 described a method for the histochemical identification of phospholipids containing choline, e.g. lecithins, lyso-lecithins, and sphingomyelins, based on their specific reaction with cis-aconitic anhydride (CAZA). The present article reports the results of an a t t e m p t to localize these phospholipids in the human aortic wall at various stages of atherosclerosis. J. Atheroscler. Res., 7 (1967) 269-281
270
E. BOELSMA-u
HOUTE, C . J . F .
BOTTCHER
MATERIALS AND METHODS
Fifteen human aortas in various stages of atherosclerosis, obtained from autopsies, were investigated histochemically. The distribution according to stage of disease and age was as follows: 5 aortas of stage 0, from individuals aged 3 and 3.5 weeks and 2.5, 5 and 14 years, 4 aortas of stage I, from 15- to 30-year-old individuals, 3 aortas of stage II, from 4O- to 60-year-old individuals, 3 aortas of stage I I I , from 40- to 60-year-old individuals. The standards used for the necropsy material and the grading of atherosclerosis* were those applied to aortas destined for chemical analysis 3. Samples were cross-sectioned on a cold microtome. The 10 # thick, frozen sections were applied to quartz slides and fixed in Baker's formol-calcium. Lipids, in particular the phospholipids, were fixed b y polymerization with a 10 ~ solution of cobaltous chloride and a 1 ~o solution of sodium periodate successively. After careful dehydration in graded alcohols and subsequent removal of the ethanol, the sections were stained for 30 rain and 1 h at room temperature in cis-aconitic anhydride (250 mg in 40 ml acetic anhydride, made up to 100 ml with toluene)l, e. To study the extractability of phospholipids in several parts of the aorta, slides were stained with CAZA for 1 h after extraction with methanol-chloroform ( 1 : 2 , b y vol.). Some of the sections were saponified with 2 N N a O H for 1 h at 37~ and then treated with CAZA for 1 h to stain sphingomyelins separately 5. Parallel sections were stained for lipids with Sudan IV. A modification of Weigert's resorcin-fuchsin method 6 and acid orcein were used as elastic tissue stains and haematoxylin and eosin were applied to stain nuclei and cytoplasm. RESULTS
The normal intima The apparently undiseased intima contained only a small amount of CAZApositive phospholipids. Most of the lecithins and/or sphingomyelins were localized in smooth muscle cells (Fig. 1) in both the very young aortas and the normal parts of the stage I aortas. The older stage 0 aorta (from a 14-year-old boy) contained foam cells showing a weak CAZA-positive reaction. In stage I the intercellular substance was slightly CAZA-positive. The phospholipids of the smooth muscle cells were mostly extractable with methanol-chloroform, although not always completely. The reaction with CAZA of the foam cells and the intercellular substance was completely negative after extraction. * Classification recommended b y the World Health Organization4: Stage O: no lesions discernible at a magnification of 10; Stage I: f a t t y streaks and spots present; Stage II: f a t t y streaks and spots accompanied b y fibrous plaques and/or atheromas; Stage III: lesions as above with additional complications, such as ulceration, necrosis, haemorrhage, calcification.
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Fig. 1. Aorta, stage 0, from a 2.S-year-old female. In the intima CAZA-positive smooth muscle cells; the fragmentated and duplicated internal elastic membrane is very Saintly stained. • 2700. The internal elastic membrane The internal elastic m e m b r a n e is composed of four intertwined elastic fibres, embedded in a matrix. This helical structure and the coating layer were sometimes visible. F r a g m e n t a t i o n and duplication start at an early age; t h e y were observed in the 2.5-year-old aorta and were c o m m o n in the older aortas, even of stage 0. The fragments and duplications also showed a helical structure. Generally, in the more diseased parts of stage I I and I I I aortas the original internal elastic m e m b r a n e could not be recognized because only fragments were left. In the y o u n g e r aortas the m e m b r a n e was slightly sudanophilic, but in a few cases of older aortas the m e m b r a n e or its fragments were more strongly stained b y Sudan IV. Phospholipids containing choline did not seem to be an integral constituent of the internal elastic m e m b r a n e : (a) in the v e r y y o u n g aortas t h e y were entirely absent, (b) in the 2.5-year-old aorta t h e y were stained only after 1 h in CAZA, (c) t h e y were not found in the aorta of a 5-year-old girl, b u t were (d) present in the stage 0 aorta of a 14-year-old boy, (e) lecithins and/or sphingomyelins were observed in one aorta of stage I (Fig. 2), and were (f) present in one of stage II. The CAZA-positive phospholipids could not be completely extracted with m e t h a n o l chloroform. After saponification the internal elastic m e m b r a n e was considerably less CAZA-positive. j . Atheroscler. Res., 7 (1967) 269-281
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Fig. 2. Aorta, stage I, from a 30-year-old female. Internal elastic membrane with CAZA-positivc phospholipids also seen locally in the coating layer, x 1750.
Fig. 3. Aorta, stage III, from a 61-year-old male. Foam ceils in a fatty streak only very faintly CAZA-positive. • 1750.
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Fatty streaks and spots The f a t t y streaks and spots showed only minor amounts of phospholipids containing choline. These lipids were found in foam cells as well as in connective tissue fibres (if present, Fig, 3). In some cases the area of the lesions bordering on the lumen of the vessel contained CAZA-positive cells. A number of f a t t y streaks contained basal cells whose small spherical nuclei reacted more positively to CAZA than the nuclei of the foam cells. Although only a few foam cells were but weakly CAZA-positive, m a n y of them showed a strong sudanophilic reaction. The intercellular substance was stainable with Sudan IV but never reacted to CAZA. The border of the fatty streaks, to the contrary, was never Sudan-positive but sometimes CAZA-positive. The phospholipids of the marginal cells were not easily extracted with methanolchloroform; extraction of the CAZA-positive lipids of the f a t t y streak contents was better, if not always complete. After saponification, sections of f a t t y streaks were not stainable with CAZA. No histochemical differences were found between the f a t t y streaks in the various stages of atherosclerosis. Morphologically, they differed only in that the stage I I and I I I aortas showed more connective tissue.
Plaques The plaques varied from completely fibrotic lesions without a histologically undifferentiated lipid mass to lesions with only a relatively thin fibrous capsule and a great deal of this so-called lipid porridge. The distribution of phospholipids containing choline was closely related to the morphological units. In the fibrous capsule CAZA-positive lipids were found in foam cells (macrophages, Fig. 4), and extracellularly, in the connective tissue fibres. Usually, the undifferentiated lipid mass was uniformly stained, though only faintly. Accumulations of phospholipids containing choline were found around cholesterol crystals (Fig. 5). Where calcareous deposits were present, these particles were also rimmed b y more intensely CAZA-positive material (Figs. 6 and 7). At the basis of the plaque degenerated elastic lamellae of the media, stained with the cis-aconitic anhydride reagent, were sometimes found. In all kinds of plaques, whether fibrotic or not, the luminal margin showed a positive reaction to CAZA locally (Fig. 8). The strongly stained lipids were situated within cells. The phospholipids containing choline from all parts of tile plaques were partially extractable with methanol-chloroform. After saponification, the sections were virtually CAZA-negative. Sudanophilic lipids were not found in the luminal part of the plaque, whereas the rest of the fibrous capsule and the lipid porridge stained strongly with Sudan IV. In general, it can be said that the reaction of the plaques to CAZA was rrmch stronger than that of the f a t t y streaks and spots. Plaques from stage n and stage I I I aortas did l~ot show any differences.
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Fig. 4. Aorta, stage II, from a 58-year-old male. CAZA-positive ceils (macrophages) in the fibrous capsule of a plaque. • 1750.
Fig. 5. Aorta, stage III, from a 61-year-old male. Lipid mass of a plaque containing cholesterol needles, surrounded by phospholipids containing choline. • 175.
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Fig. 6. Aorta, stage III, from a 61-year-old male. Calcareous deposits in a p r e d o m i n a n t l y fibrous plaque, enclosed b y CAZA-positive phospholipids. • 175.
Fig. 7. Aorta, stage III, from a 6I-year-old male, Detail of t h e plaque s h o w n in Fig. 6. • 1750.
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Fig. 8. Aorta, stage III, from a 61-year-old male. Phospholipids containing choline in the margin of a plaque. • 1750.
A theroma
One atheroma of a stage I I aorta was investigated. Only a minor amount of phospholipids containing choline was observed (Fig. 9), being located in a few nuclei, around some cholesterol needles, and in degenerated elastic lamellae of the media at the basis of the atheroma; this in spite of the fact that the extracellular lipids reacted strongly Sudan-positive. E x t r a - a n d intracellular lipids of the thin fibrous covering of the lesion were also strongly Sudan-positive but completely CAZA-negative. The small amount of CAZA-positive lipids present could be completely extracted with methanol-chloroform. The media
The most striking phenomenon in the media was the CAZA-positive reaction of the elastic lamellae (Figs. 10 and 11). Only in the very young aortas, a few weeks old, did the elastic lamellae show no reaction or only a very weak one (Fig. 12). In all other cases they were coloured homogeneously violet by the cis-aconitic anhydride reagent. Furthermore, phospholipids containing choline were found in smooth muscle cells. The CAZA-positive smooth muscle cells were easily distinguishable, especially in the very young aortas (Fig. 12). The older aortas, from stage 0 (one of a 14-year-old boy) onwards contained cells with more or less irregularly shaped nuclei; these cells showed a strong CAZA-positive reaction. After extraction with methai~oI chloroform, the elastic lamellae of aortas in all j . Atheroscler. Res., 7 (1967) 269 281
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Fig. 9. Aorta, s t a g e II, f r o m a 58-year-old male. P a r t of a n a t h e r o m a , a l m o s t n e g a t i v e l y stained; at t h e basis of t h e lesion, a c c u m u l a t i o n s of p h o s p h o l i p i d s c o n t a i n i n g choline, o r i g i n a t i n g f r o m elastic lamellae of t h e media, x 175.
Fig. I0. Aorta, s t a g e 0, f r o m a 14-year-old male, Elastic lamellae of t h e m e d i a a n d t h e i n t e r n a l elastic m e m b r a n e C A Z A - p o s i t i v e ; h a r d l y a n y r e a c t i o n in t h e i n t i m a . • 450.
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Fig. 11. Aorta, stage 0, from a 14-year-old male. Phospholipids containing choline in the elastic lamellae of the media. • 1750.
Fig. 12. Aorta, stage 0, from a 3.5-week-old female. Positively stained smooth muscle cells in the media;
hardly any reaction to CAZA
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of the elastic lamellae and of the intima.
•
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IN T H E A O R T I C W A L L
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stages of atherosclerosis reacted almost or completely negatively to CAZA. The smooth muscle cells, on the other hand, often did not even show a decreased CAZA positivity. The cells with irregularly shaped nuclei were more susceptible to extraction. The effect of saponification was not quite clear; mostly, it caused a decreased CAZA positivity in the elastic lamellae and there was no relation to the grade of atherosclerosis. The smooth muscle cells stained as strongly as before saponification. DISCUSSION
F r o m a histochemical point of view our results can be compared with those of ADAMS AND BAYLISS5. These authors applied their OTAN methods for phospholipids and for sphingomyelins to normal and atherosclerotic aortas. Chemical analyses 7 had already shown that the percentage of sphingomyelins in the phospholipids of intima plus media increases with increasing severity of atherosclerosis. BOTTCHER et al. s found 40-50 ~o sphingomyelins in the total amount of phospholipids present in the apparently normal parts of the intima, and in the media as well, from stage I I and I I I aortas. In concentric layers from stage I I and I I I aortas, with the exception of the innermost intima, ABDULLA AND ADAMS9 found 50-60 ~o sphingomyelins of the total amount of phospholipids. According to ADAMS AND BAYLISS5 0 T A N - p o s i t i v e lipids are localized in the elastic lamellae of the media - - from birth on 1~ - - and at the basis of the developing plaque. After staining with CAZA the positive reaction of the media was likewise a remarkable phenomenon. Like ADAMS AND BAYLISS, at the basis of the plaque we found a positive reaction for lipids originating from degenerated elastic lamellae. The reaction to CAZA of the smooth muscle cells from both intima and media, however, differed from that to OTAN, the former being intensely positive, the latter only weak or negative. In a more recent paper also ABDULLA AND ADAMS 9 reported the presence of phospholipids in the smooth muscle cells of the media. In our histochemical investigations the distribution of sphingomyelins did not come to the fore quite clearly, because of the precarious results especially in the media - - after saponification. ADAMS AND BAYLISS5 found nearly all the phospholipids being resistant to alkaline hydrolysis and accordingly, consisting mainly of sphingomyelins. In connection with this discrepancy reference is made to the observations of AYER 11 about destruction of the elastica in dilute alkali. We believe that this could have caused a loss of lipids from our material. In the normal intima of the very young individuals the smooth muscle cells were the only elements with phospholipids containing choline. Therefore, it is believed that these phospholipids are a normal constituent of the smooth muscle cells and that the synthesis of phospholipids in the aortic wall, demonstrated by ZILVERSMIT AND McCANDLESS 12, is accomplished b y the smooth muscle cells. On the basis of electron microscopical observations of aggregations of lipids in the smooth muscle cellslS, 14 and the fact t h a t smooth muscle cells are the only type of cell in the very young aortas 15, it is thought possible that foam cells originate from smooth muscle cells. J. Atheroscler. Res., 7 (1967) 269-281
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Since foam cells show only a weak CAZA-positivity, it seems likely that the transformation ot the smooth muscle cells into foam cells is attended b y or is the result of a disturbed phospholipid synthesis. BALIS et al. 16 observed two distinct types of foam cells, namely those derived from smooth muscle cells and, in the more advanced f a t t y streaks, those derived from macrophages. They assumed that macrophages phagocytose lipids from disintegrated myogenic foam cells, thus in turn becoming foam cells. The development of foam cells from macrophages in these aortas from 20- to 60-year-old individuals does not conflict with the mere presence of myogenic foam cells in the aortas of younger individuals, if we leave the origin of the macrophages undecided. Local accumulations of foam cells give rise to the macroscopically observable fatty streaks. The cells at the basis of the f a t t y streaks, which show a more intense CAZA positivity, could represent an intermediate stage between smooth muscle cells and foam cells. These cells are comparable to the histocytes described b y HAUST AND MORE13 as the type of cell occurring in the very early fatty streaks together with the smooth muscle cells. MCGILL AND GEER 14 described the more advanced i a t t y streaks as differing from the early ones by, among other things, the presence of extracellular lipids and more connective tissue fibres. They suppose that these lesions represent the transition form between fatty streaks and plaques. In this stage of atherosclerosis other mechanisms besides a disturbed lipid metabolism play a role: there is imbibition of lipids from the blood plasma 14, which m a y be the cause 17 of connective tissue proliferation 14, and gross fibrin formation is. In our investigations some of the f a t t y streaks showed a CAZA-positive reaction in the marginal aIea. The fact t h a t this was seen consistently in the plaques, together with the increased amount of connective tissue in these fatty streaks, leads us to assume that such lesions represent the transition form described b y MCGILL AND GEER 14. Extension at the basis of the fatty streak m a y occur b y an increase of myogenic foam cells. In a later stage of atherosclerosis the elastic lamellae of the media contribute to the formation of the so-called lipid porridge of the plaque. Within this pool of lipids, cholesterol needles and calcareous deposits are surrounded b y phospholipids containing choline. This m a y be the manifestation of the dispersion of cholesterol b y phospholipids, suggested b y ZILVERSMITAND M c C A N D L E S S 12 a s preventing the sclerogenic action of cholesterol. The latter supposition has been proved by ADAMS et al. 17 through subcutaneous injection of cholesterol and of mixtures of cholesterol and phospholipids. The former led to sclerotic lesions, whereas these failed to develop after injection of mixtures of cholesterol and phospholipids. Thus, it is conceivable that f a t t y streaks become plaques via the marginal and the basal processes described above. If, for some reason, the marginal process fails to develop, an atheroma will be formed. Histochemically, the single case under investigation completely resembled a f a t t y streak with a CAZA negative reaction in the marginal area; the basal part was identical to the lipid porridge and the degenerated elastic lamellae in plaques. j . Atheroscler. Res., 7 (1967) 269-281
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ACKNOWLEDGEMENTS T h e f i n a n c i a l s u p p o r t of t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , B e t h e s d a , Md. U . S . A . , G r a n t N o . H E 05766 ( M E T ) a n d o f t h e N e t h e r l a n d s O r g a n i z a t i o n f o r H e a l t h R e s e a r c h TNO, are gratefully acknowledged. T h a n k s a r e d u e t o Dr. J . A. v a n M e l s e n f o r h e l p f u l d i s c u s s i o n s .
REFERENCES 1 B6TTCHER, C. J. F. AND E. BOELSMA-VANHOUT~, J. Atheroseler. Res.,1964, 4: 109. 2 BOELSMA-VANHOUTE, ]~., Histochemie van Fosfolipiden in Verband met Atherosklerose van de Aorta, Thesis, Leiden, 1965. a BOTTCHER, C. J. F., F. P. WOODFORD, C. CH. TER HAAR ROMENY-WACHTER, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Lancet, 1960, i: 1378. 4 WORLD HEALTH ORGAN., Tech. Rep. Set. No. 143, 1958. s ADAMS, C. W. M. AND O. B. BAYLISS, J, Pathol. Bacteriol., 1963, 85: 113. s LAWSON W . H., J. Techn. Meth. Bull. Intern. Assoc. Med. Mus., 1936, 16: 42. 7 ]~STTCHER, C. J. ]~'. AND C. M. VAN GENT, l " Atheroscler. Res., 1961, 1: 36. s ]36TTCHER, C. J. F., et al., to be published. 9 ABDULLA, Y. H. AND C. W. M. ADAMS, J. Atheroscler. Res., 1965, 5: 504. 10 ADAMS,C. W. M. in: D. G. CHALMERSAND G. A. GRESHAM (Eds.), BiologicalAspects of Occlusive Vascular Disease, Cambridge University Press, London, 1964, p. 134. 11 AYER, J. P., in: D. A. HALL (Ed.), International Review of Connective Tissue Research, Vol.2, Academic Press, New York and London, 1964, p. 40. 12 ZILVERSMIT, D. B. AND E. L. MCCANDLESS, J. Lipid Res., 1959, 1: 118. 13 HAUST, M. D. AND R. H. MORE, in: R. J. JONES (Ed.), Evolution of the Atherosclerotic Plaque, University of Chicago Press, Chicago, 1963, p. 5I. 14 McGILL, H. C, AND J. C. GEER, in: R. J. JoNEs (Ed.), Evolution of the Atherosclerotic Plaque, University of Chicago Press, Chicago, 1963, p. 65. 15 FRENCH, J. E. in: D. G. CHALMERS AND G. A. GRESHAM (Eds.), Biological Aspects of Occlusive Vascular Disease, Cambridge University Press, London, 1964, p. 24. 16 BALIS, J. U., M. D. HAUST AND R. H. MORE, Exptl. Mol. Pathol., 1964, 3: 511. 17 ADAMS, C. W. M., O. S. BAYLISS, M. Z. M. IBRAHIM AND M. W. WEBSTER, JR., J. Pathol. Bacteriol., 1963, 86: 431. 18 HAUST, M. D., J. c. WYLLIE AND R. H. MORE, Exptl. Mol. Pathol., 1965, 4: 205.
j . Atheroscler. Res., 7 (1967) 269-281
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands L O C A L I Z A T I O N IN T H E HUMAN AORTIC W A L L OF P H O S P H O L I P I D S CONTAINING CHOLINE E. BOELSMA-VAN H O U T E AND C. J. F. BOTTCHER
Gaubius Institute of the University of Leyden, Leyden (The Netherlands) (Received September 26th, 1966)
SUMMARY
Qualitative histochemical investigations showed the following distribution of phospholipids containing choline in human aortas: (1) In the undiseased stage 0 aortas a positive reaction with cisaconitic anhydride was observed in: (a) smooth muscle cells of the intima and (b) smooth muscle cells and the elastic lamellae of the media. Positive reactions were sometimes seen in: (a) foam cells of the intima and (b) the internal elastic membrane. (2) In stage I aortas phospholipids containing choline were found in: (a) smooth muscle cells and foam cells in the intima and (b) foam cells in f a t t y streaks, (c) smooth muscle cells and elastic lamellae of the media. Occasional positive reactions were seen in: (a) the intercellular substance of the intima and (b) the internal elastic membrane. (3) In stage I I aortas phospholipids containing choline were found: (a) in foam cells of the f a t t y streaks, (b) in plaques intracellularly in the luminal margin, in myogenic foam cells (macrophages), and in connective tissue fibres, evenly spread in the undifferentiated mass of lipids with accumulations around cholesterol needles and deposits of calcium salts, in degenerated elastic lamellae, (c) in the media in smooth muscle cells and in the irregularly shaped nuclei as well as in the elastic lamellae. (4) The distribution in stage I I I aortas was identical to that in aortas of stage II; complicated lesions were not investigated. Some hypothetical statements about the origin and development of atherosclerotic lesions are discussed.
INTRODUCTION Previous papersl, 2 described a method for the histochemical identification of phospholipids containing choline, e.g. lecithins, lyso-lecithins, and sphingomyelins, based on their specific reaction with cis-aconitic anhydride (CAZA). The present article reports the results of an a t t e m p t to localize these phospholipids in the human aortic wall at various stages of atherosclerosis. J. Atheroscler. Res., 7 (1967) 269-281
270
E. BOELSMA-u
HOUTE, C . J . F .
BOTTCHER
MATERIALS AND METHODS
Fifteen human aortas in various stages of atherosclerosis, obtained from autopsies, were investigated histochemically. The distribution according to stage of disease and age was as follows: 5 aortas of stage 0, from individuals aged 3 and 3.5 weeks and 2.5, 5 and 14 years, 4 aortas of stage I, from 15- to 30-year-old individuals, 3 aortas of stage II, from 4O- to 60-year-old individuals, 3 aortas of stage I I I , from 40- to 60-year-old individuals. The standards used for the necropsy material and the grading of atherosclerosis* were those applied to aortas destined for chemical analysis 3. Samples were cross-sectioned on a cold microtome. The 10 # thick, frozen sections were applied to quartz slides and fixed in Baker's formol-calcium. Lipids, in particular the phospholipids, were fixed b y polymerization with a 10 ~ solution of cobaltous chloride and a 1 ~o solution of sodium periodate successively. After careful dehydration in graded alcohols and subsequent removal of the ethanol, the sections were stained for 30 rain and 1 h at room temperature in cis-aconitic anhydride (250 mg in 40 ml acetic anhydride, made up to 100 ml with toluene)l, e. To study the extractability of phospholipids in several parts of the aorta, slides were stained with CAZA for 1 h after extraction with methanol-chloroform ( 1 : 2 , b y vol.). Some of the sections were saponified with 2 N N a O H for 1 h at 37~ and then treated with CAZA for 1 h to stain sphingomyelins separately 5. Parallel sections were stained for lipids with Sudan IV. A modification of Weigert's resorcin-fuchsin method 6 and acid orcein were used as elastic tissue stains and haematoxylin and eosin were applied to stain nuclei and cytoplasm. RESULTS
The normal intima The apparently undiseased intima contained only a small amount of CAZApositive phospholipids. Most of the lecithins and/or sphingomyelins were localized in smooth muscle cells (Fig. 1) in both the very young aortas and the normal parts of the stage I aortas. The older stage 0 aorta (from a 14-year-old boy) contained foam cells showing a weak CAZA-positive reaction. In stage I the intercellular substance was slightly CAZA-positive. The phospholipids of the smooth muscle cells were mostly extractable with methanol-chloroform, although not always completely. The reaction with CAZA of the foam cells and the intercellular substance was completely negative after extraction. * Classification recommended b y the World Health Organization4: Stage O: no lesions discernible at a magnification of 10; Stage I: f a t t y streaks and spots present; Stage II: f a t t y streaks and spots accompanied b y fibrous plaques and/or atheromas; Stage III: lesions as above with additional complications, such as ulceration, necrosis, haemorrhage, calcification.
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Fig. 1. Aorta, stage 0, from a 2.S-year-old female. In the intima CAZA-positive smooth muscle cells; the fragmentated and duplicated internal elastic membrane is very Saintly stained. • 2700. The internal elastic membrane The internal elastic m e m b r a n e is composed of four intertwined elastic fibres, embedded in a matrix. This helical structure and the coating layer were sometimes visible. F r a g m e n t a t i o n and duplication start at an early age; t h e y were observed in the 2.5-year-old aorta and were c o m m o n in the older aortas, even of stage 0. The fragments and duplications also showed a helical structure. Generally, in the more diseased parts of stage I I and I I I aortas the original internal elastic m e m b r a n e could not be recognized because only fragments were left. In the y o u n g e r aortas the m e m b r a n e was slightly sudanophilic, but in a few cases of older aortas the m e m b r a n e or its fragments were more strongly stained b y Sudan IV. Phospholipids containing choline did not seem to be an integral constituent of the internal elastic m e m b r a n e : (a) in the v e r y y o u n g aortas t h e y were entirely absent, (b) in the 2.5-year-old aorta t h e y were stained only after 1 h in CAZA, (c) t h e y were not found in the aorta of a 5-year-old girl, b u t were (d) present in the stage 0 aorta of a 14-year-old boy, (e) lecithins and/or sphingomyelins were observed in one aorta of stage I (Fig. 2), and were (f) present in one of stage II. The CAZA-positive phospholipids could not be completely extracted with m e t h a n o l chloroform. After saponification the internal elastic m e m b r a n e was considerably less CAZA-positive. j . Atheroscler. Res., 7 (1967) 269-281
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Fig. 2. Aorta, stage I, from a 30-year-old female. Internal elastic membrane with CAZA-positivc phospholipids also seen locally in the coating layer, x 1750.
Fig. 3. Aorta, stage III, from a 61-year-old male. Foam ceils in a fatty streak only very faintly CAZA-positive. • 1750.
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Fatty streaks and spots The f a t t y streaks and spots showed only minor amounts of phospholipids containing choline. These lipids were found in foam cells as well as in connective tissue fibres (if present, Fig, 3). In some cases the area of the lesions bordering on the lumen of the vessel contained CAZA-positive cells. A number of f a t t y streaks contained basal cells whose small spherical nuclei reacted more positively to CAZA than the nuclei of the foam cells. Although only a few foam cells were but weakly CAZA-positive, m a n y of them showed a strong sudanophilic reaction. The intercellular substance was stainable with Sudan IV but never reacted to CAZA. The border of the fatty streaks, to the contrary, was never Sudan-positive but sometimes CAZA-positive. The phospholipids of the marginal cells were not easily extracted with methanolchloroform; extraction of the CAZA-positive lipids of the f a t t y streak contents was better, if not always complete. After saponification, sections of f a t t y streaks were not stainable with CAZA. No histochemical differences were found between the f a t t y streaks in the various stages of atherosclerosis. Morphologically, they differed only in that the stage I I and I I I aortas showed more connective tissue.
Plaques The plaques varied from completely fibrotic lesions without a histologically undifferentiated lipid mass to lesions with only a relatively thin fibrous capsule and a great deal of this so-called lipid porridge. The distribution of phospholipids containing choline was closely related to the morphological units. In the fibrous capsule CAZA-positive lipids were found in foam cells (macrophages, Fig. 4), and extracellularly, in the connective tissue fibres. Usually, the undifferentiated lipid mass was uniformly stained, though only faintly. Accumulations of phospholipids containing choline were found around cholesterol crystals (Fig. 5). Where calcareous deposits were present, these particles were also rimmed b y more intensely CAZA-positive material (Figs. 6 and 7). At the basis of the plaque degenerated elastic lamellae of the media, stained with the cis-aconitic anhydride reagent, were sometimes found. In all kinds of plaques, whether fibrotic or not, the luminal margin showed a positive reaction to CAZA locally (Fig. 8). The strongly stained lipids were situated within cells. The phospholipids containing choline from all parts of tile plaques were partially extractable with methanol-chloroform. After saponification, the sections were virtually CAZA-negative. Sudanophilic lipids were not found in the luminal part of the plaque, whereas the rest of the fibrous capsule and the lipid porridge stained strongly with Sudan IV. In general, it can be said that the reaction of the plaques to CAZA was rrmch stronger than that of the f a t t y streaks and spots. Plaques from stage n and stage I I I aortas did l~ot show any differences.
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Fig. 4. Aorta, stage II, from a 58-year-old male. CAZA-positive ceils (macrophages) in the fibrous capsule of a plaque. • 1750.
Fig. 5. Aorta, stage III, from a 61-year-old male. Lipid mass of a plaque containing cholesterol needles, surrounded by phospholipids containing choline. • 175.
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Fig. 6. Aorta, stage III, from a 61-year-old male. Calcareous deposits in a p r e d o m i n a n t l y fibrous plaque, enclosed b y CAZA-positive phospholipids. • 175.
Fig. 7. Aorta, stage III, from a 6I-year-old male, Detail of t h e plaque s h o w n in Fig. 6. • 1750.
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Fig. 8. Aorta, stage III, from a 61-year-old male. Phospholipids containing choline in the margin of a plaque. • 1750.
A theroma
One atheroma of a stage I I aorta was investigated. Only a minor amount of phospholipids containing choline was observed (Fig. 9), being located in a few nuclei, around some cholesterol needles, and in degenerated elastic lamellae of the media at the basis of the atheroma; this in spite of the fact that the extracellular lipids reacted strongly Sudan-positive. E x t r a - a n d intracellular lipids of the thin fibrous covering of the lesion were also strongly Sudan-positive but completely CAZA-negative. The small amount of CAZA-positive lipids present could be completely extracted with methanol-chloroform. The media
The most striking phenomenon in the media was the CAZA-positive reaction of the elastic lamellae (Figs. 10 and 11). Only in the very young aortas, a few weeks old, did the elastic lamellae show no reaction or only a very weak one (Fig. 12). In all other cases they were coloured homogeneously violet by the cis-aconitic anhydride reagent. Furthermore, phospholipids containing choline were found in smooth muscle cells. The CAZA-positive smooth muscle cells were easily distinguishable, especially in the very young aortas (Fig. 12). The older aortas, from stage 0 (one of a 14-year-old boy) onwards contained cells with more or less irregularly shaped nuclei; these cells showed a strong CAZA-positive reaction. After extraction with methai~oI chloroform, the elastic lamellae of aortas in all j . Atheroscler. Res., 7 (1967) 269 281
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Fig. 9. Aorta, s t a g e II, f r o m a 58-year-old male. P a r t of a n a t h e r o m a , a l m o s t n e g a t i v e l y stained; at t h e basis of t h e lesion, a c c u m u l a t i o n s of p h o s p h o l i p i d s c o n t a i n i n g choline, o r i g i n a t i n g f r o m elastic lamellae of t h e media, x 175.
Fig. I0. Aorta, s t a g e 0, f r o m a 14-year-old male, Elastic lamellae of t h e m e d i a a n d t h e i n t e r n a l elastic m e m b r a n e C A Z A - p o s i t i v e ; h a r d l y a n y r e a c t i o n in t h e i n t i m a . • 450.
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Fig. 11. Aorta, stage 0, from a 14-year-old male. Phospholipids containing choline in the elastic lamellae of the media. • 1750.
Fig. 12. Aorta, stage 0, from a 3.5-week-old female. Positively stained smooth muscle cells in the media;
hardly any reaction to CAZA
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of the elastic lamellae and of the intima.
•
1750.
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IN T H E A O R T I C W A L L
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stages of atherosclerosis reacted almost or completely negatively to CAZA. The smooth muscle cells, on the other hand, often did not even show a decreased CAZA positivity. The cells with irregularly shaped nuclei were more susceptible to extraction. The effect of saponification was not quite clear; mostly, it caused a decreased CAZA positivity in the elastic lamellae and there was no relation to the grade of atherosclerosis. The smooth muscle cells stained as strongly as before saponification. DISCUSSION
F r o m a histochemical point of view our results can be compared with those of ADAMS AND BAYLISS5. These authors applied their OTAN methods for phospholipids and for sphingomyelins to normal and atherosclerotic aortas. Chemical analyses 7 had already shown that the percentage of sphingomyelins in the phospholipids of intima plus media increases with increasing severity of atherosclerosis. BOTTCHER et al. s found 40-50 ~o sphingomyelins in the total amount of phospholipids present in the apparently normal parts of the intima, and in the media as well, from stage I I and I I I aortas. In concentric layers from stage I I and I I I aortas, with the exception of the innermost intima, ABDULLA AND ADAMS9 found 50-60 ~o sphingomyelins of the total amount of phospholipids. According to ADAMS AND BAYLISS5 0 T A N - p o s i t i v e lipids are localized in the elastic lamellae of the media - - from birth on 1~ - - and at the basis of the developing plaque. After staining with CAZA the positive reaction of the media was likewise a remarkable phenomenon. Like ADAMS AND BAYLISS, at the basis of the plaque we found a positive reaction for lipids originating from degenerated elastic lamellae. The reaction to CAZA of the smooth muscle cells from both intima and media, however, differed from that to OTAN, the former being intensely positive, the latter only weak or negative. In a more recent paper also ABDULLA AND ADAMS 9 reported the presence of phospholipids in the smooth muscle cells of the media. In our histochemical investigations the distribution of sphingomyelins did not come to the fore quite clearly, because of the precarious results especially in the media - - after saponification. ADAMS AND BAYLISS5 found nearly all the phospholipids being resistant to alkaline hydrolysis and accordingly, consisting mainly of sphingomyelins. In connection with this discrepancy reference is made to the observations of AYER 11 about destruction of the elastica in dilute alkali. We believe that this could have caused a loss of lipids from our material. In the normal intima of the very young individuals the smooth muscle cells were the only elements with phospholipids containing choline. Therefore, it is believed that these phospholipids are a normal constituent of the smooth muscle cells and that the synthesis of phospholipids in the aortic wall, demonstrated by ZILVERSMIT AND McCANDLESS 12, is accomplished b y the smooth muscle cells. On the basis of electron microscopical observations of aggregations of lipids in the smooth muscle cellslS, 14 and the fact t h a t smooth muscle cells are the only type of cell in the very young aortas 15, it is thought possible that foam cells originate from smooth muscle cells. J. Atheroscler. Res., 7 (1967) 269-281
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Since foam cells show only a weak CAZA-positivity, it seems likely that the transformation ot the smooth muscle cells into foam cells is attended b y or is the result of a disturbed phospholipid synthesis. BALIS et al. 16 observed two distinct types of foam cells, namely those derived from smooth muscle cells and, in the more advanced f a t t y streaks, those derived from macrophages. They assumed that macrophages phagocytose lipids from disintegrated myogenic foam cells, thus in turn becoming foam cells. The development of foam cells from macrophages in these aortas from 20- to 60-year-old individuals does not conflict with the mere presence of myogenic foam cells in the aortas of younger individuals, if we leave the origin of the macrophages undecided. Local accumulations of foam cells give rise to the macroscopically observable fatty streaks. The cells at the basis of the f a t t y streaks, which show a more intense CAZA positivity, could represent an intermediate stage between smooth muscle cells and foam cells. These cells are comparable to the histocytes described b y HAUST AND MORE13 as the type of cell occurring in the very early fatty streaks together with the smooth muscle cells. MCGILL AND GEER 14 described the more advanced i a t t y streaks as differing from the early ones by, among other things, the presence of extracellular lipids and more connective tissue fibres. They suppose that these lesions represent the transition form between fatty streaks and plaques. In this stage of atherosclerosis other mechanisms besides a disturbed lipid metabolism play a role: there is imbibition of lipids from the blood plasma 14, which m a y be the cause 17 of connective tissue proliferation 14, and gross fibrin formation is. In our investigations some of the f a t t y streaks showed a CAZA-positive reaction in the marginal aIea. The fact t h a t this was seen consistently in the plaques, together with the increased amount of connective tissue in these fatty streaks, leads us to assume that such lesions represent the transition form described b y MCGILL AND GEER 14. Extension at the basis of the fatty streak m a y occur b y an increase of myogenic foam cells. In a later stage of atherosclerosis the elastic lamellae of the media contribute to the formation of the so-called lipid porridge of the plaque. Within this pool of lipids, cholesterol needles and calcareous deposits are surrounded b y phospholipids containing choline. This m a y be the manifestation of the dispersion of cholesterol b y phospholipids, suggested b y ZILVERSMITAND M c C A N D L E S S 12 a s preventing the sclerogenic action of cholesterol. The latter supposition has been proved by ADAMS et al. 17 through subcutaneous injection of cholesterol and of mixtures of cholesterol and phospholipids. The former led to sclerotic lesions, whereas these failed to develop after injection of mixtures of cholesterol and phospholipids. Thus, it is conceivable that f a t t y streaks become plaques via the marginal and the basal processes described above. If, for some reason, the marginal process fails to develop, an atheroma will be formed. Histochemically, the single case under investigation completely resembled a f a t t y streak with a CAZA negative reaction in the marginal area; the basal part was identical to the lipid porridge and the degenerated elastic lamellae in plaques. j . Atheroscler. Res., 7 (1967) 269-281
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ACKNOWLEDGEMENTS T h e f i n a n c i a l s u p p o r t of t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , B e t h e s d a , Md. U . S . A . , G r a n t N o . H E 05766 ( M E T ) a n d o f t h e N e t h e r l a n d s O r g a n i z a t i o n f o r H e a l t h R e s e a r c h TNO, are gratefully acknowledged. T h a n k s a r e d u e t o Dr. J . A. v a n M e l s e n f o r h e l p f u l d i s c u s s i o n s .
REFERENCES 1 B6TTCHER, C. J. F. AND E. BOELSMA-VANHOUT~, J. Atheroseler. Res.,1964, 4: 109. 2 BOELSMA-VANHOUTE, ]~., Histochemie van Fosfolipiden in Verband met Atherosklerose van de Aorta, Thesis, Leiden, 1965. a BOTTCHER, C. J. F., F. P. WOODFORD, C. CH. TER HAAR ROMENY-WACHTER, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Lancet, 1960, i: 1378. 4 WORLD HEALTH ORGAN., Tech. Rep. Set. No. 143, 1958. s ADAMS, C. W. M. AND O. B. BAYLISS, J, Pathol. Bacteriol., 1963, 85: 113. s LAWSON W . H., J. Techn. Meth. Bull. Intern. Assoc. Med. Mus., 1936, 16: 42. 7 ]~STTCHER, C. J. ]~'. AND C. M. VAN GENT, l " Atheroscler. Res., 1961, 1: 36. s ]36TTCHER, C. J. F., et al., to be published. 9 ABDULLA, Y. H. AND C. W. M. ADAMS, J. Atheroscler. Res., 1965, 5: 504. 10 ADAMS,C. W. M. in: D. G. CHALMERSAND G. A. GRESHAM (Eds.), BiologicalAspects of Occlusive Vascular Disease, Cambridge University Press, London, 1964, p. 134. 11 AYER, J. P., in: D. A. HALL (Ed.), International Review of Connective Tissue Research, Vol.2, Academic Press, New York and London, 1964, p. 40. 12 ZILVERSMIT, D. B. AND E. L. MCCANDLESS, J. Lipid Res., 1959, 1: 118. 13 HAUST, M. D. AND R. H. MORE, in: R. J. JONES (Ed.), Evolution of the Atherosclerotic Plaque, University of Chicago Press, Chicago, 1963, p. 5I. 14 McGILL, H. C, AND J. C. GEER, in: R. J. JoNEs (Ed.), Evolution of the Atherosclerotic Plaque, University of Chicago Press, Chicago, 1963, p. 65. 15 FRENCH, J. E. in: D. G. CHALMERS AND G. A. GRESHAM (Eds.), Biological Aspects of Occlusive Vascular Disease, Cambridge University Press, London, 1964, p. 24. 16 BALIS, J. U., M. D. HAUST AND R. H. MORE, Exptl. Mol. Pathol., 1964, 3: 511. 17 ADAMS, C. W. M., O. S. BAYLISS, M. Z. M. IBRAHIM AND M. W. WEBSTER, JR., J. Pathol. Bacteriol., 1963, 86: 431. 18 HAUST, M. D., J. c. WYLLIE AND R. H. MORE, Exptl. Mol. Pathol., 1965, 4: 205.
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