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The relationship of arterial macrophages to the phospholipid content in rabbit atheroma
350
JOURNAL OF ATHEROSCLEROSIS RESEARCH
THE RELATIONSHIP OF ARTERIAL MACROPHAGES TO THE PHOSPHOLIPID CONTENT IN RABBIT ATHEROMA A.
J. DAY
Department of Human Physiology and Pharmacology, University of Adelaide, Adelaide (South Australia)
(Received June 6th, 1962)
INTRODUCTION Although there is a considerable literature on the morphology of atherosclerotic lesions in cholesterol-fed rabbits 1 ,2,3, no investigation has been reported of the histological distribution of phospholipid in such lesions. The phospholipid content of the aorta increases in cholesterol-fed rabbits 4 and there is now evidence that this phospholipid arises by synthesis in situ rather than by filtration from the blood5 ,6. Information regarding the cellular distribution of phospholipid in the lesions might be expected therefore to demonstrate which cells are involved in this synthesis. In the present experiments the presence and distribution of phospholipid in arterial lesions in cholesterol-fed rabbits was investigated both in aortic lesions arising as a result of the cholesterol feeding alone and in carotid lesions arising following heat injury in the cholesterol-fed rabbits. METHODS
Experimental procedure Rabbits (0.8--1.3 kg) were fed a diet containing 0.75 g cholesterol, 4.5 ml peanut oil, and 75 g rabbit mash daily for periods up to 4 months. Two rabbits were killed at each of various intervals ranging from 2 weeks to 4 months after the commencement of cholesterol feeding. One week prior to killing, the endothelium of the right carotid artery was injured by injection into the artery of 2 to 3 ml of saline at 55°C as described by COURTICE AND SCHMIDT-DIEDRICHS7. When the animal was killed the thoracic aorta and right carotid artery were removed, the aortic lesions examined and graded macroscopically as described by DAY AND WILKINSON8, and the two vessels prepared for frozen section. In a second series of cholesterol-fed rabbits, right carotid artery injection was carried out 2 weeks after commencement of the cholesterol feeding. Two rabbits were killed at various intervals from 3 days to 8 weeks after the initial carotid injury and their right carotid arteries examined.
Histological procedure Tissues were fixed in 4% formol saline containing 1% calcium chloride and both ]. Atheroscler. Res., 2 (1962) 350-364
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the aorta and injured carotid artery were cut in series as near as possible to serial section so that adjacent sections could be examined by different histochemical procedures. The sections were stained either with Sudan IV and haematoxylin, by the Schultz reaction for cholesterol9 or by Baker's acid-haematin method for phospholipids 10 . In addition, some sections were stained with Fettrot and Light Green.
Serum lipid determinations Serum cholesterol was determined by the method of ZLATKIS, ZAK AND BOYLEll and serum phospholipid by the method of BROWN12 at intervals during feeding and immediately prior to killing. RESULTS The elevation of serum lipids and the degree of macroscopic atheroma present in the cholesterol-fed rabbits used for the histological investigation are shown in Table I. Both the serum cholesterol and serum phospholipid rose rapidly from initial levels of 85 (± 5.6) mg/lOG ml and 174 (± 14) mg/lOG ml respectively to the values shown in Table I. Macroscopic lesions were first observed 2 weeks after commencement of feeding, but their occurrence was somewhat variable at this stage. Microscopic lesions were observed in all animals studied.
Aortic lesions Early lesions were characterised by the appearance of phospholipid together with cholesterol and sudanophilic material in "foam cells" in the intima. Figs. 1-3 show these "foam cell" lesions in the intima in rabbit E4 which had been fed cholesterol for 2 weeks. The distribution of the phospholipid follows essentially that of the other lipid components in the early lesion, apparently being taken up with cholesterol and triglyceride by macrophages in the subendothelial space. In more advanced lesions the distribution of phospholipid is still similar to that of cholesterol and neutral fat. Figs. 4-6 show typical intimal lesions arising after 6 weeks cholesterol feeding (rabbit E9). At this stage considerable quantities of phospholipid are present in the intima but it is still possible to see that the phospholipid remains intracellularly in the macrophages which comprise the main bulk of the intimal lesion. After 15 weeks cholesterol feeding, the intimal lesions show proliferative changes. The relative amount of sudanophilic material and cholesterol has diminished and some of it is present extracellularly at the base of the intimal lesions adjacent to the internal elastic lamina. The phospholipid however remains distributed intracellularly in macrophages scattered throughout the now organising lesion (Figs. 7-10).
Carotid artery lesions Injury by heat and trauma in the cholesterol-fed rabbits resulted in intimal thickening and lipid infiltration of both intima and media (Figs. 11-14). It is apparent that tissue macrophages, mobilised following the infiltration, have taken up phospholipid in addition to sudanophilic material and cholesterol, so that one week after (Continuedonp.361)
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Fig. 1. Early atherosclerotic lesion showing phospholipid intracellularly in rnacrophages. Rabbit aorta after 2 weeks cholesterol feeding. Baker acid-haernatin; x 165.
Fig. 2. Adjacent section to Fig. 1. Fettrot and Light Green; :< 165.
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Fig. 3. Adjacent section to Figs. 1, 2. Schultz; x 165.
Fig. 4. Atherosclerotic lesion in rabbit aorta after 6 weeks cholesterol feeding showing phospholipid present intracellu1arly in the intimal macrophages. Baker acid-haematin; X llO.
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Fig. 5. Adjacent section to Fig. 4. Sudan IV; X 110.
Fig. 6. Adjacent section to Fig. 4. Schultz;
X
110.
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Fig. 7. Advanced atherosclerotic lesion in rabbit aorta after 15 weeks cholesterol feeding. The phospholipid present in the intima11esion is confined to the macrophages scattered throughout the area. Baker acid-haematin; X 110.
Fig. 8. Adjacent section to Fig. 7. Sudan IV; X 110.
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Fig. 9. Adjacent section to Fig. 7. Schultz; X 110.
Fig. 10. Same section as Fig. 7. Baker acid-haematin;
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Fig. 11. Right carotid artery 7 days after intimal damage b» heat. Rabbit fed cholesterol for 3 weeks. Infiltration of media has occurred and phospholipid has been taken up by mobilised macrophages. Baker acid-haematin; X 110.
Fig. 12. Adjacent section to Fig. 11. Sudan IV; X 110. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 13. Adjacent section to Fig. 11. Schultz; y. 110.
Fig. 14. Right carotid artery 7 days after intimal damage by heat. Rabbit fed cholesterol for 3 weeks. Intimal thickening with lipid infiltration can be seen. Sudan IV; x 110. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 15. Same section as Fig. 11. Baker acid-hacmatin; ./ 445.
Fig. 16. Same section as Fig. 12. Sudan IV; X 445. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 17. Right carotid artery 7 weeks after intimal damage by heat. Cholesterol-fed rabbit. Phospholipid has almost disappeared from the intimal thickening which has developed. Baker acid-haematin; X 45.
Fig. 18. Adjacent section to Fig. 17. Sudan IV; x 45.
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Fig. 19. Adjacent section to Fig. 17. Schultz; X 45.
Injury the media contains considerable amounts of phospholipid, sudanophilic material and cholesterol, all present intracellularly in mobilised macrophages (Figs. 15, 16).
Removal of lipid following carotid injury In the second series of cholesterol-fed rabbits, lesions in the right carotid artery TABLE I SERUM
LIPID
LEVELS
AND
MACROSCOPIC
ATHEROMA
PRESENT AT
VARIOUS
INTERVALS
AFTER
COMMENCEMENT OF CHOLESTEROL FEEDING IN THE RABBITS USED FOR HISTOLOGICAL INVESTIGATION
Rabbit No.
Weeks of feeding
E4 E12 E5 Ell E7 E14 El E9 E3 ElO E2 E6 E13
2 2 3 3 4 4 6 6 8 8 12 15 15
Serum lipid prior to death (mg/l00 ml) Cholesterol
A theroma grade
Phospholipid
1420 1680 1840 2130 1880 2870 1890 2160 3760 2700* 2630* 1540** 2920**
790 910 635 1000 875 925 750 750 1325 800* 925* 550** 1160**
0 I 0 0 I 0 I II IV III III 0 IV
* Determined at 5 weeks. ** Determined at 6 weeks.
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were allowed to progress for various intervals from 3 days to 8 weeks after injury. Initial lesions were similar to those described above in the first series of rabbits. Four to eight weeks after injury, lipid had been removed from the intimal lesions leaving some intimal thickening with only a few scattered macrophages containing lipid (Figs. 17-19). The degree of removal of lipid, however, was not always the same which wa::; largely due to variable injury and it was not possible to ascertain whether any difference occurred in removal of total fat or cholesterol from that of phospholipid. DISCUSSION
The Baker acid-haematin method is considered specific, among lipids, for phospholipid 9 . Baker-positive material was present only in areas showing positive Sudan IV staining and appeared only in the intimal macrophages in the atheromatous arterial wall, and it is known that the phospholipid content of the arterial wall increases as a result of cholesterol feeding 4,5. There seems little doubt therefore that what has been demonstrated in these experiments as Baker-positive material in the macrophages is phospholipid. In the very early microscopic lesions where only a few lipid-filled macrophages are in evidence, phospholipid is present intracellularly in the macrophages in addition to cholesterol and triglyceride. Thus all the lipid components of the lipoprotein are taken up by these cells, and this fact is consistent with the conclusion that lipoprotein is taken up unchanged by macrophages in the arterial wall. This conclusion is supported by recent work 13 where it has been shown that 3H-cholesterol labelled lipoprotein obtained from cholesterol-fed rabbits can be taken up by rabbit macrophages in vitro. BUCK14 has postulated on electron microscopic evidence that lipoprotein, filtered through the endothelial wall in the cholesterol-fed rabbit, has its protein removed prior to ingestion of its lipid by the macrophages. If this were so one might expect some dispersion of the lipid, the more soluble phospholipid being removed and the insoluble cholesterol and triglyceride portion being taken up by macrophages. The presence of phospholipid in macrophages in the early aortic lesions may be partly due to synthesis in the cells. Yet, where sudden lipid infiltration of the arterial wall has occurred following carotid injury, phospholipid as well as cholesterol and sudanophilic material can still be observed intracellularly in the arterial wall. This implies that phospholipid in lipoprotein filtered from the plasma can be actively taken up by tissue macrophages accumulated around the lesions. Recently the part played by metabolism in the arterial wall in the pathogenesis of atherosclerosis has come into prominence. ZILVERSMIT et al. 5 ,15 have demonstrated that phospholipid is actively synthesised in the arterial wall in both experimental and human atheroma. In the present observations rabbits with equivalent lesions to those induced by these workers have been shown to contain large amounts of phospholipid, all of which is contained intracellularly in the intimal macrophages. It seems reasonable therefore to infer that these cells are responsible for the phospholipid synthesis which occurs in the atheromatous arterial wall. The observation that
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phospholipid can be synthesised by macrophages in vitro using either 14C-Iabelled fatty acid 16 or 14C-Iabelled acetate 17 as precursors support this conclusion. It would seem that much of the phospholipid metabolism of atherosclerotic lesions is explicable in terms of metabolism of their contained macrophages. Since macrophages are known to influence cholesterol metabolism 18 ,19 also, it appears probable that lipid metabolism by macrophages is a major factor responsible for the metabolism of lipid known to occur in atheroma in the arterial wall. ACKNOWLEDGEMENTS
This work was aided by a grant from the National Heart Foundation of Australia. I am indebted to Misses E. WADLOW, H. WILSON and M. KLEEMAN for their technical assistance. SUMMARY
The cellular distribution of phospholipid in aortic lesions in cholesterol-fed rabbits was investigated by the Baker acid-haematin method for phospholipid, and compared in adjacent sections with the distribution of cholesterol and non-specific sudanophilic material. Over a wide range of severity of aortic lesions, phospholipid was shown to be present entirely intracellularly in intimal macrophages indicating that the synthesis of phospholipid known to take place in the arterial wall in atherosclerosis occurs as a result of metabolism by the mobilised macrophages. In carotid arterial lesions produced by heat injury in cholesterol-fed rabbits, phospholipid (along with cholesterol and sudanophilic material) infiltrated the intima and media and was readily taken up intracellularly by mobilised macrophages. Removal of the phospholipid from the macrophages over a period of 8 weeks following the initial injury was observed but it was not possible to determine whether phospholipid was removed from the macrophages more readily than other lipid components. The metabolism by macrophages in the arterial wall and their significance in the pathogenesis of atherosclerosis are discussed.
La distribution cellulaire des phospholipides dans les lesions aortiques de lapins nourris au cholesterol est etudiee par la methode de Baker a I'Mmatine acide et comparee avec la distribution du cholesterol et du materiel soudanophile non specifique. Dans les lesions aortiques de gravite tres differente, les phospholipides sont trouves entierement dans les macrophages de l'intima. Ce fait indique que la synthese des phospholipides dans la paroi arterielle atMrosclereuse est Ie resultat du metabolisme des macrophages. Dans les lesicns de l'artere carotide produites par brulures chez les lapins nourris au cholesterol, les phospholipides (ainsi que Ie cholesterol et Ie materiel soudanophile) j. Atheroscler. Res., 2 (1962) 350-364
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infiltrent l'intima et la media et sont rapidement phagocytes par les macrophages mobilises. La disparition des phospholipides contenus dans les macrophages fut observee au cours d'une periode de huit semaines suivant Ie traumatisme initial mais il ne fut pas possible de preciser si les phospholipides disparaissaient plus vite que les autres composants lipidiques. Le role des macrophages dans Ie metabolisme de la paroi arterielle et sa signification dans la pathogenie de l'atherosclerose sont discutes. ZUSAMMENFASSUNG
Mit Hilfe der Baker'schen Saure-Haematinmethode zum Nachweis von Phospholipid wurde bei cholesteringeftitterten Kaninchen die zellulare Verteilung des Phospholipids in den Aortenlasionen untersucht und in Serienschnitten mit der Verteilung des Cholesterins und des unspezifischen sudanophilen Materials verglichen. Es zeigte sich, dass bei den verschiedensten Schweregraden der Aortenveranderungen Phospholipid ausschliesslich intrazellular in den Intimamakrophagen liegt. Daraus geht hervor, dass die Synthese des Phospholipids, die bekanntlich bei der Atherosklerose in der Arterienwand stattfindet, ein Stoffwechseleffekt mobilisierter Makrophagen darstellt. In den Carotidenlasionen, die bei cholesteringefiitterten Kaninchen durch Hitzeeinwirkung erzeugt wurden, infiltrierte Phospholipid (zusammen mit Cholesterin und sudanophilem Material) die Intima und Media und wurde rasch intrazellular von mobilisierten Makrophagen aufgenommen. Wahrend 8 Wochen nach der initialen Schadigung wurde eine Entfernung von Phospholipid aus den Makrophagen beobachtet, aber es war nicht moglich festzustellen, ob Phospholipid schneller als andere Lipidkomponenten aus den Makrophagen entfernt wurde. Es wird der Anteil des Stoffwechsels der Makrophagen in der Arterienwand und ihre Bedeutung in der Pathogenese der Atherosklerose besprochen. REFERENCES 1 2 3
4 5 6
7 8 9
10 11
12 13 14
15 16
17 18
19
G. L. DUFF, A.M.A. Arch. Pathol., 20 (1935) 81, 259. R. ALTSCHUL, Selected Studies on Atherosclerosis, C. C. Thomas, Springfield, Ill., 1950. G. L. McMILLAN, 1. KLATZO AND G. L. DUFF, Lab. Invest., 3 (1954) 451. S. WEINHOUSE AND E. F. HIRSCH, A. M. A. Arch. Pathol., 30 (1940) 856. M. L. SHORE, D. B. ZILVERSMIT AND R. F. ACKERMAN, Am. ]. Physiol., 181 (1955) 527. D. B. ZILVERSMIT AND E. L. MCCANDLESS, J. Lipid Research, 1 (1959) 118. F. C. COURTICE AND A. SCHMIDT-DIEDRICHS, Quart. ]. Exptl. Physiol., 47 (1962) 228. A. J. DAY AND G. K. WILKINSON, Aust. J. Exptl. Bioi., 34 (1956) 423. A. G. E. PEARSE, Histochemistry, Theoretical and Applied, 2nd ed., Churchill Ltd., London, 1960. J. R. BAKER, Quart. ]. Micr. Sci., 87 (1946) 441. A. ZLATKIS, B. ZAK AND A. J. BOYLE, ]. Lab. Clin. l\Ied., 41 (1953) 486. W. D. BROWN, Aust. ]. Exptl. Bioi., 32 (1954) 677. A. J. DAY AND M. L. WAHLQVIST, unpublished data. R. C. BUCK, Am. ]. Pathol., 34 (1958) 897. D. B. ZILVERSMIT, E. L. MCCANDLESS, P. H. JORDAN, W. S. HENLEY AND R. F. ACKERMAN, Circulation, 23 (1961) 370. A. J. DAY AND N. H. FIDGE, ]. Lipid Research, 3 (1962) 333. A. J. DAY AND N. H. FIDGE, Unpublished data. A. J. DAY, Quart. ]. Exptl. Physiol., 45 (1960) 55. A. J. DAY AND P. R. S. GOULD-HuRST, Quart. ]. Exptl. Physiol., 46 (1961) 376.
.f. .,ltheroscler. Res., 2 (1962) 350-364
JOURNAL OF ATHEROSCLEROSIS RESEARCH
THE RELATIONSHIP OF ARTERIAL MACROPHAGES TO THE PHOSPHOLIPID CONTENT IN RABBIT ATHEROMA A.
J. DAY
Department of Human Physiology and Pharmacology, University of Adelaide, Adelaide (South Australia)
(Received June 6th, 1962)
INTRODUCTION Although there is a considerable literature on the morphology of atherosclerotic lesions in cholesterol-fed rabbits 1 ,2,3, no investigation has been reported of the histological distribution of phospholipid in such lesions. The phospholipid content of the aorta increases in cholesterol-fed rabbits 4 and there is now evidence that this phospholipid arises by synthesis in situ rather than by filtration from the blood5 ,6. Information regarding the cellular distribution of phospholipid in the lesions might be expected therefore to demonstrate which cells are involved in this synthesis. In the present experiments the presence and distribution of phospholipid in arterial lesions in cholesterol-fed rabbits was investigated both in aortic lesions arising as a result of the cholesterol feeding alone and in carotid lesions arising following heat injury in the cholesterol-fed rabbits. METHODS
Experimental procedure Rabbits (0.8--1.3 kg) were fed a diet containing 0.75 g cholesterol, 4.5 ml peanut oil, and 75 g rabbit mash daily for periods up to 4 months. Two rabbits were killed at each of various intervals ranging from 2 weeks to 4 months after the commencement of cholesterol feeding. One week prior to killing, the endothelium of the right carotid artery was injured by injection into the artery of 2 to 3 ml of saline at 55°C as described by COURTICE AND SCHMIDT-DIEDRICHS7. When the animal was killed the thoracic aorta and right carotid artery were removed, the aortic lesions examined and graded macroscopically as described by DAY AND WILKINSON8, and the two vessels prepared for frozen section. In a second series of cholesterol-fed rabbits, right carotid artery injection was carried out 2 weeks after commencement of the cholesterol feeding. Two rabbits were killed at various intervals from 3 days to 8 weeks after the initial carotid injury and their right carotid arteries examined.
Histological procedure Tissues were fixed in 4% formol saline containing 1% calcium chloride and both ]. Atheroscler. Res., 2 (1962) 350-364
ARTERIAL MACROPHAGES AND THE PHOSPHOLIPID CONTENT IN RABBIT ATHEROMA
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the aorta and injured carotid artery were cut in series as near as possible to serial section so that adjacent sections could be examined by different histochemical procedures. The sections were stained either with Sudan IV and haematoxylin, by the Schultz reaction for cholesterol9 or by Baker's acid-haematin method for phospholipids 10 . In addition, some sections were stained with Fettrot and Light Green.
Serum lipid determinations Serum cholesterol was determined by the method of ZLATKIS, ZAK AND BOYLEll and serum phospholipid by the method of BROWN12 at intervals during feeding and immediately prior to killing. RESULTS The elevation of serum lipids and the degree of macroscopic atheroma present in the cholesterol-fed rabbits used for the histological investigation are shown in Table I. Both the serum cholesterol and serum phospholipid rose rapidly from initial levels of 85 (± 5.6) mg/lOG ml and 174 (± 14) mg/lOG ml respectively to the values shown in Table I. Macroscopic lesions were first observed 2 weeks after commencement of feeding, but their occurrence was somewhat variable at this stage. Microscopic lesions were observed in all animals studied.
Aortic lesions Early lesions were characterised by the appearance of phospholipid together with cholesterol and sudanophilic material in "foam cells" in the intima. Figs. 1-3 show these "foam cell" lesions in the intima in rabbit E4 which had been fed cholesterol for 2 weeks. The distribution of the phospholipid follows essentially that of the other lipid components in the early lesion, apparently being taken up with cholesterol and triglyceride by macrophages in the subendothelial space. In more advanced lesions the distribution of phospholipid is still similar to that of cholesterol and neutral fat. Figs. 4-6 show typical intimal lesions arising after 6 weeks cholesterol feeding (rabbit E9). At this stage considerable quantities of phospholipid are present in the intima but it is still possible to see that the phospholipid remains intracellularly in the macrophages which comprise the main bulk of the intimal lesion. After 15 weeks cholesterol feeding, the intimal lesions show proliferative changes. The relative amount of sudanophilic material and cholesterol has diminished and some of it is present extracellularly at the base of the intimal lesions adjacent to the internal elastic lamina. The phospholipid however remains distributed intracellularly in macrophages scattered throughout the now organising lesion (Figs. 7-10).
Carotid artery lesions Injury by heat and trauma in the cholesterol-fed rabbits resulted in intimal thickening and lipid infiltration of both intima and media (Figs. 11-14). It is apparent that tissue macrophages, mobilised following the infiltration, have taken up phospholipid in addition to sudanophilic material and cholesterol, so that one week after (Continuedonp.361)
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Fig. 1. Early atherosclerotic lesion showing phospholipid intracellularly in rnacrophages. Rabbit aorta after 2 weeks cholesterol feeding. Baker acid-haernatin; x 165.
Fig. 2. Adjacent section to Fig. 1. Fettrot and Light Green; :< 165.
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Fig. 3. Adjacent section to Figs. 1, 2. Schultz; x 165.
Fig. 4. Atherosclerotic lesion in rabbit aorta after 6 weeks cholesterol feeding showing phospholipid present intracellu1arly in the intimal macrophages. Baker acid-haematin; X llO.
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Fig. 5. Adjacent section to Fig. 4. Sudan IV; X 110.
Fig. 6. Adjacent section to Fig. 4. Schultz;
X
110.
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Fig. 7. Advanced atherosclerotic lesion in rabbit aorta after 15 weeks cholesterol feeding. The phospholipid present in the intima11esion is confined to the macrophages scattered throughout the area. Baker acid-haematin; X 110.
Fig. 8. Adjacent section to Fig. 7. Sudan IV; X 110.
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Fig. 9. Adjacent section to Fig. 7. Schultz; X 110.
Fig. 10. Same section as Fig. 7. Baker acid-haematin;
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Fig. 11. Right carotid artery 7 days after intimal damage b» heat. Rabbit fed cholesterol for 3 weeks. Infiltration of media has occurred and phospholipid has been taken up by mobilised macrophages. Baker acid-haematin; X 110.
Fig. 12. Adjacent section to Fig. 11. Sudan IV; X 110. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 13. Adjacent section to Fig. 11. Schultz; y. 110.
Fig. 14. Right carotid artery 7 days after intimal damage by heat. Rabbit fed cholesterol for 3 weeks. Intimal thickening with lipid infiltration can be seen. Sudan IV; x 110. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 15. Same section as Fig. 11. Baker acid-hacmatin; ./ 445.
Fig. 16. Same section as Fig. 12. Sudan IV; X 445. ]. Atheroscler. Res., 2 (1962) 350-364
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Fig. 17. Right carotid artery 7 weeks after intimal damage by heat. Cholesterol-fed rabbit. Phospholipid has almost disappeared from the intimal thickening which has developed. Baker acid-haematin; X 45.
Fig. 18. Adjacent section to Fig. 17. Sudan IV; x 45.
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Fig. 19. Adjacent section to Fig. 17. Schultz; X 45.
Injury the media contains considerable amounts of phospholipid, sudanophilic material and cholesterol, all present intracellularly in mobilised macrophages (Figs. 15, 16).
Removal of lipid following carotid injury In the second series of cholesterol-fed rabbits, lesions in the right carotid artery TABLE I SERUM
LIPID
LEVELS
AND
MACROSCOPIC
ATHEROMA
PRESENT AT
VARIOUS
INTERVALS
AFTER
COMMENCEMENT OF CHOLESTEROL FEEDING IN THE RABBITS USED FOR HISTOLOGICAL INVESTIGATION
Rabbit No.
Weeks of feeding
E4 E12 E5 Ell E7 E14 El E9 E3 ElO E2 E6 E13
2 2 3 3 4 4 6 6 8 8 12 15 15
Serum lipid prior to death (mg/l00 ml) Cholesterol
A theroma grade
Phospholipid
1420 1680 1840 2130 1880 2870 1890 2160 3760 2700* 2630* 1540** 2920**
790 910 635 1000 875 925 750 750 1325 800* 925* 550** 1160**
0 I 0 0 I 0 I II IV III III 0 IV
* Determined at 5 weeks. ** Determined at 6 weeks.
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were allowed to progress for various intervals from 3 days to 8 weeks after injury. Initial lesions were similar to those described above in the first series of rabbits. Four to eight weeks after injury, lipid had been removed from the intimal lesions leaving some intimal thickening with only a few scattered macrophages containing lipid (Figs. 17-19). The degree of removal of lipid, however, was not always the same which wa::; largely due to variable injury and it was not possible to ascertain whether any difference occurred in removal of total fat or cholesterol from that of phospholipid. DISCUSSION
The Baker acid-haematin method is considered specific, among lipids, for phospholipid 9 . Baker-positive material was present only in areas showing positive Sudan IV staining and appeared only in the intimal macrophages in the atheromatous arterial wall, and it is known that the phospholipid content of the arterial wall increases as a result of cholesterol feeding 4,5. There seems little doubt therefore that what has been demonstrated in these experiments as Baker-positive material in the macrophages is phospholipid. In the very early microscopic lesions where only a few lipid-filled macrophages are in evidence, phospholipid is present intracellularly in the macrophages in addition to cholesterol and triglyceride. Thus all the lipid components of the lipoprotein are taken up by these cells, and this fact is consistent with the conclusion that lipoprotein is taken up unchanged by macrophages in the arterial wall. This conclusion is supported by recent work 13 where it has been shown that 3H-cholesterol labelled lipoprotein obtained from cholesterol-fed rabbits can be taken up by rabbit macrophages in vitro. BUCK14 has postulated on electron microscopic evidence that lipoprotein, filtered through the endothelial wall in the cholesterol-fed rabbit, has its protein removed prior to ingestion of its lipid by the macrophages. If this were so one might expect some dispersion of the lipid, the more soluble phospholipid being removed and the insoluble cholesterol and triglyceride portion being taken up by macrophages. The presence of phospholipid in macrophages in the early aortic lesions may be partly due to synthesis in the cells. Yet, where sudden lipid infiltration of the arterial wall has occurred following carotid injury, phospholipid as well as cholesterol and sudanophilic material can still be observed intracellularly in the arterial wall. This implies that phospholipid in lipoprotein filtered from the plasma can be actively taken up by tissue macrophages accumulated around the lesions. Recently the part played by metabolism in the arterial wall in the pathogenesis of atherosclerosis has come into prominence. ZILVERSMIT et al. 5 ,15 have demonstrated that phospholipid is actively synthesised in the arterial wall in both experimental and human atheroma. In the present observations rabbits with equivalent lesions to those induced by these workers have been shown to contain large amounts of phospholipid, all of which is contained intracellularly in the intimal macrophages. It seems reasonable therefore to infer that these cells are responsible for the phospholipid synthesis which occurs in the atheromatous arterial wall. The observation that
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phospholipid can be synthesised by macrophages in vitro using either 14C-Iabelled fatty acid 16 or 14C-Iabelled acetate 17 as precursors support this conclusion. It would seem that much of the phospholipid metabolism of atherosclerotic lesions is explicable in terms of metabolism of their contained macrophages. Since macrophages are known to influence cholesterol metabolism 18 ,19 also, it appears probable that lipid metabolism by macrophages is a major factor responsible for the metabolism of lipid known to occur in atheroma in the arterial wall. ACKNOWLEDGEMENTS
This work was aided by a grant from the National Heart Foundation of Australia. I am indebted to Misses E. WADLOW, H. WILSON and M. KLEEMAN for their technical assistance. SUMMARY
The cellular distribution of phospholipid in aortic lesions in cholesterol-fed rabbits was investigated by the Baker acid-haematin method for phospholipid, and compared in adjacent sections with the distribution of cholesterol and non-specific sudanophilic material. Over a wide range of severity of aortic lesions, phospholipid was shown to be present entirely intracellularly in intimal macrophages indicating that the synthesis of phospholipid known to take place in the arterial wall in atherosclerosis occurs as a result of metabolism by the mobilised macrophages. In carotid arterial lesions produced by heat injury in cholesterol-fed rabbits, phospholipid (along with cholesterol and sudanophilic material) infiltrated the intima and media and was readily taken up intracellularly by mobilised macrophages. Removal of the phospholipid from the macrophages over a period of 8 weeks following the initial injury was observed but it was not possible to determine whether phospholipid was removed from the macrophages more readily than other lipid components. The metabolism by macrophages in the arterial wall and their significance in the pathogenesis of atherosclerosis are discussed.
La distribution cellulaire des phospholipides dans les lesions aortiques de lapins nourris au cholesterol est etudiee par la methode de Baker a I'Mmatine acide et comparee avec la distribution du cholesterol et du materiel soudanophile non specifique. Dans les lesions aortiques de gravite tres differente, les phospholipides sont trouves entierement dans les macrophages de l'intima. Ce fait indique que la synthese des phospholipides dans la paroi arterielle atMrosclereuse est Ie resultat du metabolisme des macrophages. Dans les lesicns de l'artere carotide produites par brulures chez les lapins nourris au cholesterol, les phospholipides (ainsi que Ie cholesterol et Ie materiel soudanophile) j. Atheroscler. Res., 2 (1962) 350-364
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infiltrent l'intima et la media et sont rapidement phagocytes par les macrophages mobilises. La disparition des phospholipides contenus dans les macrophages fut observee au cours d'une periode de huit semaines suivant Ie traumatisme initial mais il ne fut pas possible de preciser si les phospholipides disparaissaient plus vite que les autres composants lipidiques. Le role des macrophages dans Ie metabolisme de la paroi arterielle et sa signification dans la pathogenie de l'atherosclerose sont discutes. ZUSAMMENFASSUNG
Mit Hilfe der Baker'schen Saure-Haematinmethode zum Nachweis von Phospholipid wurde bei cholesteringeftitterten Kaninchen die zellulare Verteilung des Phospholipids in den Aortenlasionen untersucht und in Serienschnitten mit der Verteilung des Cholesterins und des unspezifischen sudanophilen Materials verglichen. Es zeigte sich, dass bei den verschiedensten Schweregraden der Aortenveranderungen Phospholipid ausschliesslich intrazellular in den Intimamakrophagen liegt. Daraus geht hervor, dass die Synthese des Phospholipids, die bekanntlich bei der Atherosklerose in der Arterienwand stattfindet, ein Stoffwechseleffekt mobilisierter Makrophagen darstellt. In den Carotidenlasionen, die bei cholesteringefiitterten Kaninchen durch Hitzeeinwirkung erzeugt wurden, infiltrierte Phospholipid (zusammen mit Cholesterin und sudanophilem Material) die Intima und Media und wurde rasch intrazellular von mobilisierten Makrophagen aufgenommen. Wahrend 8 Wochen nach der initialen Schadigung wurde eine Entfernung von Phospholipid aus den Makrophagen beobachtet, aber es war nicht moglich festzustellen, ob Phospholipid schneller als andere Lipidkomponenten aus den Makrophagen entfernt wurde. Es wird der Anteil des Stoffwechsels der Makrophagen in der Arterienwand und ihre Bedeutung in der Pathogenese der Atherosklerose besprochen. REFERENCES 1 2 3
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