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Distortion of endothelial repair The effect of hypercholesterolaemia on regeneration of aortic endothelium following injury by endotoxin A scanning electron microscope study
459
Atherosclerosis, 29 (1978) 459-466 0 Elsevier/North-Holland Scientific Publishers, Ltd.
DISTORTION
OF ENDOTHELIAL
REPAIR
THE EFFECT OF HYPERCHOLESTEROLAEMIA ON REGENERATION OF AORTIC ENDOTHELIUM FOLLOWING INJURY BY ENDOTOXIN A Scanning Electron Microscope Study
M.A. REIDY and D.E. BOWYER Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP (Great Britain) (Received 4 October, 1977) (Revised, received 9 December, 1977) (Accepted 9 December, 1977)
Summary
Five young male New Zealand White rabbits were fed a semi-synthetic diet containing 0.2% cholesterol for 2 weeks and a control group of 5 animals was fed a normal stock diet. All animals were then injected intravenously with a single dose of endotoxin from Serrutia marcescens (200 pg/kg body weight) and continued on their respective diets for a further 4 weeks. The aortas were then stained with silver nitrate and fixed under pressure for Scanning Electron Microscopy (SEM). Argyrophilic endothelial cells were present in both groups of animals 4 weeks after endotoxin injections. In the cholesterol-fed animals, however, these cells were often covered with pits and craters. These findings suggest that the hypercholesterolaemia may affect the regeneration of arterial endothelial cells. Key words:
Endothelial microscopy
repair - Endotoxin
- Hypercholesterolaemia
-Scanning
electron
Introduction
Endothelial injury is known to facilitate atherogenesis probably by permitting an increased entry of blood constituents into the arterial wall. Thus
The
authors
are grateful
to May
and
Baker,
Ltd.,
Dagenham
for
financial
support.
460
lipoproteins and lipids accumulate and smooth muscle cells may be stimulated to proliferate both by lipoproteins [l] and platelet constituents [ 21. In experimental animals the induction of atherosclerotic lesions following endothelial injury, depends not only upon the severity of the initial damage and the degree of hypercholesterolaemia, but also upon the speed of endothelial regeneration [3]. Thus, Bjijrkerud [4,5] has demonstrated that if endothelial regrowth is rapid, then the initial lesions may regress, whereas if regeneration of endothelial cells is slow then the ensuing lesion is more severe and persists. Many studies have examined the responses of the arterial wall to direct injury [3-131 although few have tried to study the effect of interfering with endothelial regeneration. Nam et al. [14] injured the aortas of swine with a balloon catheter and then induced hypercholesterolaemia. They found that after 3 months the sites of injury were grossly atherosclerotic. Other studies [ 15-171, however, show that hypercholesterolaemia and arterial injury do not necessarily cause a severe lesion. Thus, Clowes et al. [15] showed that, in rat arteries injured by a jet of air directed onto the endothelial surface, there is no increase in intimal thickening in the cholesterol-fed animals as compared to the animals receiving a normal diet. The purpose of this study was directly to injure arterial endothelial cells in hypercholesterolaemic rabbits and to observe the morphology of the luminal cells at various times after the initial injury. The endothelium was injured by a single intravenous injection of endotoxin which causes widespread endothelial damage [ 131. The progress of endothelial regeneration in the normal and hypercholesterolaemic animals was followed by Scanning Electron Microscopy (SEM) as previously described [ 131. Methods and Materials Ten male New Zealand White rabbits, approximately 2 kg body weight were used in this study. The animals were divided into 2 groups as follows: Group 1: Five animals were fed a normal diet for 2 weeks and then given a single i.v. injection of endotoxin P-45 Poly Serratia marcescens, a gift from Dr. D.B. Cater, (200 pg/kg body weight), suspended in sterile isotonic saline. They were killed 28 days later. Group 2: Five animals were fed a diet containing 0.2% cholesterol and 20% beef tallow for 2 weeks and then injected with endotoxin as in Group 1. The cholesterol-containing diet was continued and they were killed 28 days later. Prepare tion of aortas for SEM
The methods were essentially those described previously by us [13,18,19]. Each animal was injected via an ear vein with Heparin (200 units/kg body weight) and killed by a blow on the head. A small opening was made in the thoracic cavity, the aorta was exposed and a femoral artery severed to allow the blood to drain. A plastic cannula was inserted into the aorta through which 30 ml of 4.6% glucose solution, buffered with N-2 hydroxyethylpiperazine-N’2-ethanesulphonic acid (HEPES, 20 mM, pH 7.4) was introduced to wash out the residual blood. A 0.2% solution of silver nitrate in 4.2% glucose buffered with HEPES (20 mM, pH 7.4)) was then passed into the aorta for
461
approximately 30 sec. The aorta was flushed again with 30 ml of the 4.6% buffered glucose solution and then fixed with 2% phosphate-buffered formalin, pH 7.4. By means of a three-way tap connected to the cannula, the above solutions were infused into the aorta at a constant pressure. The femoral artery was ligated and the aorta was left in situ to fix for 18 h at an approximate pressure of 100 mm Hg. The fixed vessel was carefully excised, pinned out onto cork boards, and washed in distilled water. The tissue was then allowed to air-dry and stored in a dessicator. Results In all the animals fed cholesterol (Group 2), small fatty lesions were observed on their aortic surface. These lesions were located around the aortic ostia, but in no other regions. For the purpose of this study these areas with atherosclerotic lesions were excluded and the results presented are from those areas of the aorta where no fatty lesions were observed. Four weeks after endotoxin administration the aortas from the control-fed animals (Group 1) showed endothelial cells which appeared brightly stained when viewed by SEM. The entire surface of such cells was heavily stained with silver and they will be referred to as argyrophilic cells (Figs. 1 and 2). These argyrophilic cells were often found to form rows along the length of the aorta with normal endothelial cells around them. There were also areas which were entirely denuded of endothelium, and the edges of these regions were argyrophilic .
Fig. 1. The luminal surface of aorta of a control normolipaemic rabbit 28 days after injection with endotoxin. Silver nitrate stain, X 310. Scale bar 100 gm.
462
Fig. 2. As in Fig. 1. X770. Scale bar 40 Wm.
Fig. 3. The luminal surface of aorta of a hyperlipaemic rabbit 28 days after injection Silver nitrate stain. X260. Scale bar 100 pm.
with endotoxin.
463
Fig. 4. As in Fig. 3. X770. Scale bar 40 pm.
The luminal surface of the aortas from cholesterol-fed animals killed 4 weeks after endotoxin administration (Group 2), contained a variety of differing endothelial cells. Some small bodies and endothelial cells with bright boundaries were observed as well as argyrophilic cells similar to those in animals of Groun 1. Many argyrophilic cells, however, showed an abnormal morphology and their cell surface was covered with pits and small craters (Figs. 3 and 4). In some instances obvious signs of cellular damage were present. Normal endothelial cells were found throughout the remainder of these aortas. Regions denuded of endothelium were also observed, but despite the hyperlipaemia in these animals, no raised intimal thickening was observed at these sites. Discussion The results of this study showed that the formation of argyrophilic endothelial cells was altered in the hypercholesterolaemic animals. Four weeks after endotoxin administration the argyrophilic cells from the cholesterol-fed animals were markedly different from those found in the aortas of the controlfed animals (Group 2). These cells showed signs of cellular degeneration and their surfaces were covered with pits and small craters. It could be suggested that these breaks in cellular morphology represent arterial fatty lesions which have collapsed during the fixation and drying procedure. This is unlikely since in previous reports [18-201 we have shown that if fatty arterial lesions were air-dried without the use of organic solvents, then they did not collapse and
464
their raised structure was preserved. Furthermore, the gross appearance of the endothelial cells overlying the lesions was not altered by this drying procedure. It remains a possibility that the endothelial cells in animals with hypercholesterolaemia and injected with endotoxin, have a composition which makes them especially susceptible to the staining, fixation, drying and coating procedures. Volatile lipids might have been evaporated from the cells during sputter coating, thus leaving craters. Newly formed endothelial cells are known to be more permeable than adult cells [21,22] and in a previous study [ 131 we suggested that the argyrophilic endothelial cells observed several weeks after endotoxin injury were regenerating or young cells. Similar heavily silver-stained endothelial cells have been reported by Gottlob and Zinner [23] who considered them either newly formed cells or else injured cells which were permeable to the silver salts. Christensen [8] observed argyrophilic endothelial cells in rabbit aorta 17 days after noradrenalin injury and also concluded that they were either young regenerating cells or damaged ones. Poole et al. [9], however, noted that endothelial cells bordering denuded zones were heavily stained with silver granules and thought them to be newly formed endothelial cells and not damaged cells. In support of this hypothesis, we have recently observed [24] similar argyrophilic endothelial cells in the aortas of l-week old rabbits and also on the hips of aortic flow dividers of older animals. At both these sites endothelial cells are known to have a high mitotic index [ 25,261. The present study suggests that regenerating endothelial cells were more susceptible to the hyperlipaemia than were the surrounding normal endothelial cells. Possibly the high concentration of cholesterol in the plasma was toxic to these young cells causing them to exhibit altered morphology. Indeed Schwartz et al. [29] have shown by transmission electron microscopy that reestablished endothelial cells formed after injury have certain features not found in normal endothelium. These cells also often fail to form gap junctions between adjoining membranes. SEM studies have also shown a difference between recently regenerated and normal endothelial cells. Christensen and Garbarsch [7] reported that the endothelial cells located over a recently injured site were often abnormal in their morphology and were oriented in a different direction than the surrounding normal endothelial cells. It is interesting to note that while fatty lesions were observed distal to aortic ostia they were not observed in those regions of the aorta with endotoxininduced injury, nor in the denuded areas. While some studies. [14,30-321 report that endothelial injury combined with hypercholesterolaemia will accelerate the process of atherosclerosis, other work [ 15-171 has shown that a combination of these two treatments does not necessarily lead to an enhancement of the disease. Indeed, Clowes et al. [15] reported no differences in intimal thickening of injured arteries between normocholesterolaemic and hypercholesterolaemic rats. In a study using pigs, Nam et al. [14] found that if aortas were subjected to a combination of hypercholesterolaemia and injury then the ensuing atherosclerotic lesions were far more severe than in uninjured aortas. This difference, however, as judged both by intimal thickening and surface involvement was only really noticeable 3 months after the start of the experiment. Thus, it may be that a critical time period is required to elapse
465
before lesions form. In this study, therefore, the areas damaged by endotoxin might have been sites at which lesions would have subsequently developed had the experiment been continued. The implications of this study are that hypercholesterolaemia may distort endothelial growth and regeneration and this may be one mechanism by which this risk factor operates to cause atherosclerosis. Acknowledgements We are grateful to Mr. K. Thurley and Mr. W. Moue1 of the Electron Microscopy Unit, Department of Anatomy, University of Cambridge for their valuable help. We wish to thank Mrs. L.M. Wright for secretarial assistance and Mr. M. Pollard and Mr. R. Burgess for animal husbandry. References 1 Dzoga, K., Vesselinovitch, D., Fraser, R. and Wissler. R.W., The effect of lipoproteins on the growth of aortic smooth muscle cells in vitro, Amer. J. Path., 62 (1971) 320. 2 Ross, R.. Glomset. J., Kariya. B. and Harker, L.. A platelet-dependent serum factor that stimulates the proliferation of smooth muscle cells in vitro, Proc. Natl. Acad. Sci. (Wash.), 71 (1974) 1207. 3 BjGrkerud. S. and Bondjers. G., Arterial repair and atherosclerosis after mechanical injury, Part 5 (Tissue response after induction of a large superficial transverse injury), Atherosclerosis, 18 (1973) 235. 4 BjBrkerud, S.. Atherosclerosis initiated by mechanical trauma in normo-lipidaemic rabbits, Atherosclerosis. 9 (1969) 209. 5 Bjiirkerud. S., Reaction of the aortic wall of the rabbit after superficial longitudinal mechanical trauma, Virchows Arch. Aht. A. Path. Anat.. 347 (1969) 197. 6 Bjiirkerud, S.. Injury and repair in arterial wall in relation to injury and repair - A survey, Angiology. 25 (1974) 636. 7 Christensen, C.B. and Garbarsch. C.. Repair in arterial tissue - A scanning electron microscopic and light microscopic study on the endothelium of rabbit thoracic aorta following a single dilation injury, Virchows Arch. Abt. A. Path. Anat.. 360 (1973) 93. 8 Christensen, C.B.. Repair in arterial tissue - A SEM and light microscopic study on the endothelium of rabbit thoracic aorta following noradrenalin in toxic doses. Virchows Arch. Abt. A. Path. Anat.. 363 (1974) 33. 9 Poole, J.C.. Sanders, A.G. and Florey. H.W.. The regeneration of aortic endothelium. J. Path. Bact.. 75 (1968) 133. 10 Constantinides, P. and Robinson, M., Ultrastructural injury of arterial endothelium. Arch. Path., 88 (1969) 99. 11 Hoff, H.F., Vascular injury - A review. In: Vascular Factors and Thrombosis (Thromb. Diath. Haem. Suppl. 40). Schattauer Verlag. Stuttgart, 1970. 12 Helin. P.. Lorenzen. I., Garbarsch, C. and Matthiessen. M.E., Repsir in arterial tissue -Morphological and biochemical changes in rabbit aorta after a single dilation injury. Circulat. Res.. 29 (1971) 642. 13 Reidy, M.A. and Bowyer. D.E.. Scanning electron microscopy - Morphology of aortic endothelium following injury by endotoxin and during subsequent repsir, Atherosclerosis, 26 (1977) 319. 14 Nam, S.C., Lee. WM.. Jarmolych, J., Lee, K.T. and Thomas, W.. Rapid production of advanced atherosclerosis in swine by a combination of endothelial injury and cholesterol feeding, Exp. Mol. Path., 18 (1973) 369. 15 Clowes. A.W.. Ryan. G.B.. Breslow. J.L. and Karnovsky, M.,Abscnce of enhanced intimal thickening in the response of the carotid arterial wsB to endothelial injury in hypercholesterolaemic rats, Lab. Invest., 35 (1976) 6. 16 Wissler, R.W.. Eilert, N.L., Schroeder, M.A. and Cohen, L.. Production of lipomatous and atheromatous arterial lesions in the albino rat, Arch. Path., 67 (1964) 333. 17 Hardin. N.J., Minick. C.P. and Murphy, C.E., Experimental induction of atherosclerosis by the synergy of allergic injury to arteries and lipid rich diet, Amer. J. Path.. 73 (1973) 301. I8 Davies, P.F.. Reidy. M.A., Goode, T.B. and Bowyer. D-E.. Scanning electron microscopy -A method to evaluate endothelial integrity of the fatty lesion of atherosclerosis, Atherosclerosis. 26 (1976) 126. 19 Reidy. M.A. and Bowyer, D.E., Precise location of fatty streak lesions of atherosclerosis in experimental animals, Artery, 2 (1977) 574.
466 20 Goode, T.B.. Davies, P.F., Reidy, M.A. and Bowyer. D.E., Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit, Atherosclerosis, 21(1977) 236. 21 Adams, C.W.M. Vascular Histochemistry. Lloyd-Luke, London, 1967, p. 193. 22 Friedman, M. and Byers. S.O., Excess lipid leakage -A property of very young vascular endothelium. Brit. J. Exp. Path., 43 (1962) 363. 23 Gottlob, R. and Zinner. G., ijber die Regeneration geschadigtes Endothelium nach hartem und weichem Trauma, Virchows Arch. Abt. A. Path. Anat.. 336 (1962) 16. 24 Reidy. M.A., Unpublished results. 26 Schwartz. SM. and Benditt, E.P.. Cell replication in the aortic endothelhrm - A new method for study of the problem, Lab. Invest., 28 (1973) 699. 26 Wright, H.P., Mitosis patterns in aortic endothelium, Atherosclerosis, 16 (1972) 93. 27 Thomas, W.A.. Florentin, R.A., Nam, S.C.. Kim. D.N., Jones, R.M. and Lee, K.T.. Pre-proliferative phase of atherosclerosis in swine fed cholesterol, Arch. Path., 86 (1968) 621. 28 Trillo. A.. Renaud.S. and Haust. M.D., The morphogenesis of aortic lesions in hypertensive and hypertensive hyperlipaemic rats, Lab. Invest., 24 (1971) 461. 29 Schwartz. S.M.. Sternerman. M. and Benditt. E.. The aortic intima. Part 3 (Repair of the aortic lining after mechanical denudation), Amer. J. Path., 81 (1976) 16. 30 Lee, W.M. and Lee. K.T.. Advanced coronary atherosclerosis in swine produced by combination of balloon-catheter injury and cholesterol feeding, Exp. Mol. Path., 23 (1975) 491. 31 Koletsky, A., Roland, C. and Rivera-Velez. J., Rapid acceleration of atherosclerosis in hypertensive rats on high fat diet, Exp. Mol. Path., 9 (1968) 322. 32 Pick, R.. Johnson, P. and Glick, G.. Deleterious effects of hypertension on the development of aortic and coronary atherosclerosis in stumptail macaques (Mocaca specioso) on an atherogenic diet, Circulat. Res., 36 (1974) 412.
Atherosclerosis, 29 (1978) 459-466 0 Elsevier/North-Holland Scientific Publishers, Ltd.
DISTORTION
OF ENDOTHELIAL
REPAIR
THE EFFECT OF HYPERCHOLESTEROLAEMIA ON REGENERATION OF AORTIC ENDOTHELIUM FOLLOWING INJURY BY ENDOTOXIN A Scanning Electron Microscope Study
M.A. REIDY and D.E. BOWYER Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP (Great Britain) (Received 4 October, 1977) (Revised, received 9 December, 1977) (Accepted 9 December, 1977)
Summary
Five young male New Zealand White rabbits were fed a semi-synthetic diet containing 0.2% cholesterol for 2 weeks and a control group of 5 animals was fed a normal stock diet. All animals were then injected intravenously with a single dose of endotoxin from Serrutia marcescens (200 pg/kg body weight) and continued on their respective diets for a further 4 weeks. The aortas were then stained with silver nitrate and fixed under pressure for Scanning Electron Microscopy (SEM). Argyrophilic endothelial cells were present in both groups of animals 4 weeks after endotoxin injections. In the cholesterol-fed animals, however, these cells were often covered with pits and craters. These findings suggest that the hypercholesterolaemia may affect the regeneration of arterial endothelial cells. Key words:
Endothelial microscopy
repair - Endotoxin
- Hypercholesterolaemia
-Scanning
electron
Introduction
Endothelial injury is known to facilitate atherogenesis probably by permitting an increased entry of blood constituents into the arterial wall. Thus
The
authors
are grateful
to May
and
Baker,
Ltd.,
Dagenham
for
financial
support.
460
lipoproteins and lipids accumulate and smooth muscle cells may be stimulated to proliferate both by lipoproteins [l] and platelet constituents [ 21. In experimental animals the induction of atherosclerotic lesions following endothelial injury, depends not only upon the severity of the initial damage and the degree of hypercholesterolaemia, but also upon the speed of endothelial regeneration [3]. Thus, Bjijrkerud [4,5] has demonstrated that if endothelial regrowth is rapid, then the initial lesions may regress, whereas if regeneration of endothelial cells is slow then the ensuing lesion is more severe and persists. Many studies have examined the responses of the arterial wall to direct injury [3-131 although few have tried to study the effect of interfering with endothelial regeneration. Nam et al. [14] injured the aortas of swine with a balloon catheter and then induced hypercholesterolaemia. They found that after 3 months the sites of injury were grossly atherosclerotic. Other studies [ 15-171, however, show that hypercholesterolaemia and arterial injury do not necessarily cause a severe lesion. Thus, Clowes et al. [15] showed that, in rat arteries injured by a jet of air directed onto the endothelial surface, there is no increase in intimal thickening in the cholesterol-fed animals as compared to the animals receiving a normal diet. The purpose of this study was directly to injure arterial endothelial cells in hypercholesterolaemic rabbits and to observe the morphology of the luminal cells at various times after the initial injury. The endothelium was injured by a single intravenous injection of endotoxin which causes widespread endothelial damage [ 131. The progress of endothelial regeneration in the normal and hypercholesterolaemic animals was followed by Scanning Electron Microscopy (SEM) as previously described [ 131. Methods and Materials Ten male New Zealand White rabbits, approximately 2 kg body weight were used in this study. The animals were divided into 2 groups as follows: Group 1: Five animals were fed a normal diet for 2 weeks and then given a single i.v. injection of endotoxin P-45 Poly Serratia marcescens, a gift from Dr. D.B. Cater, (200 pg/kg body weight), suspended in sterile isotonic saline. They were killed 28 days later. Group 2: Five animals were fed a diet containing 0.2% cholesterol and 20% beef tallow for 2 weeks and then injected with endotoxin as in Group 1. The cholesterol-containing diet was continued and they were killed 28 days later. Prepare tion of aortas for SEM
The methods were essentially those described previously by us [13,18,19]. Each animal was injected via an ear vein with Heparin (200 units/kg body weight) and killed by a blow on the head. A small opening was made in the thoracic cavity, the aorta was exposed and a femoral artery severed to allow the blood to drain. A plastic cannula was inserted into the aorta through which 30 ml of 4.6% glucose solution, buffered with N-2 hydroxyethylpiperazine-N’2-ethanesulphonic acid (HEPES, 20 mM, pH 7.4) was introduced to wash out the residual blood. A 0.2% solution of silver nitrate in 4.2% glucose buffered with HEPES (20 mM, pH 7.4)) was then passed into the aorta for
461
approximately 30 sec. The aorta was flushed again with 30 ml of the 4.6% buffered glucose solution and then fixed with 2% phosphate-buffered formalin, pH 7.4. By means of a three-way tap connected to the cannula, the above solutions were infused into the aorta at a constant pressure. The femoral artery was ligated and the aorta was left in situ to fix for 18 h at an approximate pressure of 100 mm Hg. The fixed vessel was carefully excised, pinned out onto cork boards, and washed in distilled water. The tissue was then allowed to air-dry and stored in a dessicator. Results In all the animals fed cholesterol (Group 2), small fatty lesions were observed on their aortic surface. These lesions were located around the aortic ostia, but in no other regions. For the purpose of this study these areas with atherosclerotic lesions were excluded and the results presented are from those areas of the aorta where no fatty lesions were observed. Four weeks after endotoxin administration the aortas from the control-fed animals (Group 1) showed endothelial cells which appeared brightly stained when viewed by SEM. The entire surface of such cells was heavily stained with silver and they will be referred to as argyrophilic cells (Figs. 1 and 2). These argyrophilic cells were often found to form rows along the length of the aorta with normal endothelial cells around them. There were also areas which were entirely denuded of endothelium, and the edges of these regions were argyrophilic .
Fig. 1. The luminal surface of aorta of a control normolipaemic rabbit 28 days after injection with endotoxin. Silver nitrate stain, X 310. Scale bar 100 gm.
462
Fig. 2. As in Fig. 1. X770. Scale bar 40 Wm.
Fig. 3. The luminal surface of aorta of a hyperlipaemic rabbit 28 days after injection Silver nitrate stain. X260. Scale bar 100 pm.
with endotoxin.
463
Fig. 4. As in Fig. 3. X770. Scale bar 40 pm.
The luminal surface of the aortas from cholesterol-fed animals killed 4 weeks after endotoxin administration (Group 2), contained a variety of differing endothelial cells. Some small bodies and endothelial cells with bright boundaries were observed as well as argyrophilic cells similar to those in animals of Groun 1. Many argyrophilic cells, however, showed an abnormal morphology and their cell surface was covered with pits and small craters (Figs. 3 and 4). In some instances obvious signs of cellular damage were present. Normal endothelial cells were found throughout the remainder of these aortas. Regions denuded of endothelium were also observed, but despite the hyperlipaemia in these animals, no raised intimal thickening was observed at these sites. Discussion The results of this study showed that the formation of argyrophilic endothelial cells was altered in the hypercholesterolaemic animals. Four weeks after endotoxin administration the argyrophilic cells from the cholesterol-fed animals were markedly different from those found in the aortas of the controlfed animals (Group 2). These cells showed signs of cellular degeneration and their surfaces were covered with pits and small craters. It could be suggested that these breaks in cellular morphology represent arterial fatty lesions which have collapsed during the fixation and drying procedure. This is unlikely since in previous reports [18-201 we have shown that if fatty arterial lesions were air-dried without the use of organic solvents, then they did not collapse and
464
their raised structure was preserved. Furthermore, the gross appearance of the endothelial cells overlying the lesions was not altered by this drying procedure. It remains a possibility that the endothelial cells in animals with hypercholesterolaemia and injected with endotoxin, have a composition which makes them especially susceptible to the staining, fixation, drying and coating procedures. Volatile lipids might have been evaporated from the cells during sputter coating, thus leaving craters. Newly formed endothelial cells are known to be more permeable than adult cells [21,22] and in a previous study [ 131 we suggested that the argyrophilic endothelial cells observed several weeks after endotoxin injury were regenerating or young cells. Similar heavily silver-stained endothelial cells have been reported by Gottlob and Zinner [23] who considered them either newly formed cells or else injured cells which were permeable to the silver salts. Christensen [8] observed argyrophilic endothelial cells in rabbit aorta 17 days after noradrenalin injury and also concluded that they were either young regenerating cells or damaged ones. Poole et al. [9], however, noted that endothelial cells bordering denuded zones were heavily stained with silver granules and thought them to be newly formed endothelial cells and not damaged cells. In support of this hypothesis, we have recently observed [24] similar argyrophilic endothelial cells in the aortas of l-week old rabbits and also on the hips of aortic flow dividers of older animals. At both these sites endothelial cells are known to have a high mitotic index [ 25,261. The present study suggests that regenerating endothelial cells were more susceptible to the hyperlipaemia than were the surrounding normal endothelial cells. Possibly the high concentration of cholesterol in the plasma was toxic to these young cells causing them to exhibit altered morphology. Indeed Schwartz et al. [29] have shown by transmission electron microscopy that reestablished endothelial cells formed after injury have certain features not found in normal endothelium. These cells also often fail to form gap junctions between adjoining membranes. SEM studies have also shown a difference between recently regenerated and normal endothelial cells. Christensen and Garbarsch [7] reported that the endothelial cells located over a recently injured site were often abnormal in their morphology and were oriented in a different direction than the surrounding normal endothelial cells. It is interesting to note that while fatty lesions were observed distal to aortic ostia they were not observed in those regions of the aorta with endotoxininduced injury, nor in the denuded areas. While some studies. [14,30-321 report that endothelial injury combined with hypercholesterolaemia will accelerate the process of atherosclerosis, other work [ 15-171 has shown that a combination of these two treatments does not necessarily lead to an enhancement of the disease. Indeed, Clowes et al. [15] reported no differences in intimal thickening of injured arteries between normocholesterolaemic and hypercholesterolaemic rats. In a study using pigs, Nam et al. [14] found that if aortas were subjected to a combination of hypercholesterolaemia and injury then the ensuing atherosclerotic lesions were far more severe than in uninjured aortas. This difference, however, as judged both by intimal thickening and surface involvement was only really noticeable 3 months after the start of the experiment. Thus, it may be that a critical time period is required to elapse
465
before lesions form. In this study, therefore, the areas damaged by endotoxin might have been sites at which lesions would have subsequently developed had the experiment been continued. The implications of this study are that hypercholesterolaemia may distort endothelial growth and regeneration and this may be one mechanism by which this risk factor operates to cause atherosclerosis. Acknowledgements We are grateful to Mr. K. Thurley and Mr. W. Moue1 of the Electron Microscopy Unit, Department of Anatomy, University of Cambridge for their valuable help. We wish to thank Mrs. L.M. Wright for secretarial assistance and Mr. M. Pollard and Mr. R. Burgess for animal husbandry. References 1 Dzoga, K., Vesselinovitch, D., Fraser, R. and Wissler. R.W., The effect of lipoproteins on the growth of aortic smooth muscle cells in vitro, Amer. J. Path., 62 (1971) 320. 2 Ross, R.. Glomset. J., Kariya. B. and Harker, L.. A platelet-dependent serum factor that stimulates the proliferation of smooth muscle cells in vitro, Proc. Natl. Acad. Sci. (Wash.), 71 (1974) 1207. 3 BjGrkerud. S. and Bondjers. G., Arterial repair and atherosclerosis after mechanical injury, Part 5 (Tissue response after induction of a large superficial transverse injury), Atherosclerosis, 18 (1973) 235. 4 BjBrkerud, S.. Atherosclerosis initiated by mechanical trauma in normo-lipidaemic rabbits, Atherosclerosis. 9 (1969) 209. 5 Bjiirkerud. S., Reaction of the aortic wall of the rabbit after superficial longitudinal mechanical trauma, Virchows Arch. Aht. A. Path. Anat.. 347 (1969) 197. 6 Bjiirkerud, S.. Injury and repair in arterial wall in relation to injury and repair - A survey, Angiology. 25 (1974) 636. 7 Christensen, C.B. and Garbarsch. C.. Repair in arterial tissue - A scanning electron microscopic and light microscopic study on the endothelium of rabbit thoracic aorta following a single dilation injury, Virchows Arch. Abt. A. Path. Anat.. 360 (1973) 93. 8 Christensen, C.B.. Repair in arterial tissue - A SEM and light microscopic study on the endothelium of rabbit thoracic aorta following noradrenalin in toxic doses. Virchows Arch. Abt. A. Path. Anat.. 363 (1974) 33. 9 Poole, J.C.. Sanders, A.G. and Florey. H.W.. The regeneration of aortic endothelium. J. Path. Bact.. 75 (1968) 133. 10 Constantinides, P. and Robinson, M., Ultrastructural injury of arterial endothelium. Arch. Path., 88 (1969) 99. 11 Hoff, H.F., Vascular injury - A review. In: Vascular Factors and Thrombosis (Thromb. Diath. Haem. Suppl. 40). Schattauer Verlag. Stuttgart, 1970. 12 Helin. P.. Lorenzen. I., Garbarsch, C. and Matthiessen. M.E., Repsir in arterial tissue -Morphological and biochemical changes in rabbit aorta after a single dilation injury. Circulat. Res.. 29 (1971) 642. 13 Reidy, M.A. and Bowyer. D.E.. Scanning electron microscopy - Morphology of aortic endothelium following injury by endotoxin and during subsequent repsir, Atherosclerosis, 26 (1977) 319. 14 Nam, S.C., Lee. WM.. Jarmolych, J., Lee, K.T. and Thomas, W.. Rapid production of advanced atherosclerosis in swine by a combination of endothelial injury and cholesterol feeding, Exp. Mol. Path., 18 (1973) 369. 15 Clowes. A.W.. Ryan. G.B.. Breslow. J.L. and Karnovsky, M.,Abscnce of enhanced intimal thickening in the response of the carotid arterial wsB to endothelial injury in hypercholesterolaemic rats, Lab. Invest., 35 (1976) 6. 16 Wissler, R.W.. Eilert, N.L., Schroeder, M.A. and Cohen, L.. Production of lipomatous and atheromatous arterial lesions in the albino rat, Arch. Path., 67 (1964) 333. 17 Hardin. N.J., Minick. C.P. and Murphy, C.E., Experimental induction of atherosclerosis by the synergy of allergic injury to arteries and lipid rich diet, Amer. J. Path.. 73 (1973) 301. I8 Davies, P.F.. Reidy. M.A., Goode, T.B. and Bowyer. D-E.. Scanning electron microscopy -A method to evaluate endothelial integrity of the fatty lesion of atherosclerosis, Atherosclerosis. 26 (1976) 126. 19 Reidy. M.A. and Bowyer, D.E., Precise location of fatty streak lesions of atherosclerosis in experimental animals, Artery, 2 (1977) 574.
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