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Spontaneous arterial lesions: Their possible role in arteriosclerosis
EXPERIMENTAL
AND
MOLECULAR
Spontaneous
PATHOLOGY
45, 227-244 (1986)
Arterial Lesions: Their in Arteriosclerosis
Possible
Role
GORDON R. CAMPBELL AND PETER R. L. MOSSE Department
of Anatomy, Received
University
Noijember
Il.
of Melbourne.
Parkville,
1985, and in revisedform
3052,
Victoria.
April
22. 1986
Australia
Spontaneous lesions of the arterial wall involving the internal elastic lamellae and variable amounts of the intima are described in the spontaneously hypertensive rat caudal. renal, and mesenteric arteries. A simple model for producing similar circumferential lesions in rat and rabbit carotid arteries has been developed and the subsequent repair of these lesions is described. Two types of circumferential lesion can be produced by the application of 50- 160 g of longitudinally applied tension. Small lesions can be up to 400 km in length and are characterized by the loss of a small area of endothelium and rupture of the internal elastic lamellae. No demonstrable damage to the media is detected in these lesions. Larger lesions can be up to 1 mm in length and are characterized by the loss of endothelium and rupture of the internal as well as a variable number of medial elastic lamellae. Little, if any, damage to the medial smooth muscle ceils is observed although the extracellular matrix is often disrupted. Small lesions are completely reendothelialized within 24 hr and larger lesions within 7-10 days. Both large and small lesions repair without the formation of an intimal thickening of smooth muscle cells, despite quite marked damage to the media of the larger lesions. 0 1986 Academic Press. Inc.
INTRODUCTION The major components of the aortic media are smooth muscle cells, elastic lamellae, collagen, and proteoglycans, which together form complex, integrated functional units (Glagov, 1979). Throughout the vascular system the contribution of each of these components with its differing properties varies according to the functional demands placed upon the particular vessel. The relationship between components is dynamic in nature with the unit capable of remodelling its structure to accommodate any change in demands, as for instance in growth and hypertension (see Campbell and Charnley-Campbell, 1981). Changes within one component of the vessel wall, whether normal or pathological, affects others. One function of the elastic lamellae of arteries is to distribute the tensile stresses uniformly throughout the wall (Glagov, 1979). Any dramatic change in these lamellae, such as fragmentation or loss of structural integrity leads to a localized concentration of stress at this site. Depending on the size of the lesion and condition of the vessel wall at that point, a sudden increase in pressure may in turn lead to pathological consequences. In 1979 Osborne-Pellegrin reported “spontaneous” lesions in the caudal artery of mature male Wistar rats. These lesions consisted of regions where the internal elastic lamellae (IEL) was absent over all or part of the vessel circumference. In some cases after rupture the lamellae together with the overlying endothelial cells had curled back along the vessel. Further studies by the same authors suggested flow rate and growth were important factors in the formation of these lesions (Osborne-Pellegrin ef al., 1980; Osborne-Pellegrin and Weill, 1983). In 1980 we reported a similar phenomenon in rat renal arteries (Campbell and Campbell, 227 0014-4800/86 $3.00 Copyright k5 1986 hy Academic Press, Inc. All rIghI* of reproduction in any form reserved.
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1980) and these observations have been later contirmed by Osborne-Pellegrin and co-workers (Coutard and Osborne-Pellegrin, 1982). The present article describes “spontaneous” lesions and their apparent consequences in the renal and mesenteric arteries of old hypertensive rats. A model is also described for producing similar lesions in rat and rabbit carotid artery, and the repair mechanisms following such damage examined. These data are compared with reports of repair to “spontaneous” injury (Osborne-Pellegrin and Weill, 1983). MATERIALS Part 1: Spontaneous
AND METHODS
Lesions
Caudal, renal, and mesenteric arteries of 4- to 18-month-old Kyoto-Wistar spontaneously hypertensive (SHR) (blood pressures > 180 mm Hg), and normotensive male rats were examined in this study. The renal artery was examined in two regions, just proximal to the suprarenal branch and at the bifurcation into the smaller renal arteries, close to the hilus of the kidney. A variety of mesenteric arteries of differing sizes was chosen for examination. Sixty-three SHR and 46 normotensive animals were used. The rats were anesthetized with ether, their heart exposed, and a cannula inserted into the left ventricle. The left jugular vein was exposed and cut at the commencement of perfusion. After initial perfusion washout with 0.1 It4 phosphate buffer, pH 7.3, the animal was fixed with 5% glutaraldehyde in 0.1 M phosphate buffer at pressure approximating that in situ for 15 min. The above arteries were quickly dissected free and further fixed for 1 hr, washed overnight in buffer, and postfixed in 1% osmium tetroxide for 1 hr. Part 2: Experimental Model Sixteen male New Zealand white rabbits (1.5-2.5 kg) and 21 female and 3 male Sprague-Dawley rats (200-250 g) were used. Rabbits were maintained on a daily ration of 100 g of pellet food with greens once a week. Rats were allowed free access to pellet food. Experimental procedure. Prior to surgery rats were anesthetized with ether; rabbits were preanesthetized with Epontal (Bayer Australia), intubated and maintained on Halothane (ICI Australia). In both rats and rabbits the left and right common carotid arteries were exposed by a mid-ventral incision in the neck. The arteries were cleared of surrounding connective tissue and nerves and two double loop ties were passed around the artery. These ties were separated by a distance of approximately 1 cm in rats and 1 to 1.5 cm in rabbits. Longitudinal tension was applied via these loops and vessel wall integrity monitored through x 2 operating glasses. At the first sign of damage, tension was released and the ties quickly removed. The amount of tension required to produce damage varied between animals, therefore no attempt was made to use a uniform tension each time. Between 50 and 160 g tension was required to produce the lesion in both rats and rabbits. The animals were closed and the rabbits were given an intramuscular injection of the antibiotic Reverin (Rolitetracycline 50 mg/kg, Hoechst). Fixation. Two rabbits were examined at each of 3.5-hr, l-day, 3-day, 5-day, 7-day, IO-day, 5-week, and IO-week time intervals. Three rats were examined at each of 2-hr, 3-day, 3-week, 6-week, 12-week, and 20-week intervals. At the 3week time interval, three females and three males were examined for comparison.
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Prior to perfusion fixation rats were anesthetized with ether and rabbits with intravenous sodium pentobarbitol. The abdominal aorta was exposed and a retrograde cannula introduced. The left jugular vein was exposed and cut at the start of perfusion. The animals were perfused with Hanks’ balanced salt solution (CSL, Melbourne, Australia) until the perfusate from the vein was clear of blood. The animal was then fixed with 5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, at high pressure for 5- 10 min. Both carotids were quickly removed and immersed in fixative for a further 1 hr, washed in 0.1 M phosphate buffer, pH 7.3, and postfixed in 1% osmium tetroxide for 1 hr. Only areas between the two ties were examined since in some cases the ties caused trauma to the vessel wall. Tissue for scanning electron microscopy was subsequently dehydrated in a graded ethanol series, critical point dried with carbon dioxide, and gold coated in an Edwards sputter coater. Specimens were viewed in an Etec Autoscan scanning electron microscope at 30 kV. Light and transmission electron microscopy. Tissue for light and transmission electron microscopy was dehydrated in a graded acetone series and embedded in Epon/Araldite. Thick sections were cut on glass knives and stained with methylene blue. Thin sections were cut on diamond knives, stained with uranyl acetate and lead citrate and viewed either in a Siemens or a Philips EM400 electron microscope at 60 kV. RESULTS Part I: Spontaneous
Lesions
Spontaneous lesions in rat caudal and renal arteries have been described in detail in previous reports (Wexler, 1964, 1970; Osborne-Pellegrin, 1979; Campbell and Campbell, 1980; Osborne-Pellegrin and Weill, 1983). Therefore only a brief description will be provided for comparison with the experimental lesions described in Part 2. A typical lesion in the SHR caudal artery is shown in Fig. 1. Here, the IEL is absent over a region of approximately 100 km and folded back at the edges as if it has snapped under tension. The area between the edges is reendothelialized and the media has in this case been little affected by the lamellar changes. Lesions similar to those described in renal and caudal arteries were also observed in a range of different sized mesenteric arteries. Since these lesions also appear to develop randomly, different stages of repair could be observed in different regions, even of the same artery. A number of lesions appeared to have been reendothelialized some time previously as they were covered with apparently normal endothelium (Figs. 2,6). However, the most common lesions were those which appeared to have been reendothelialized recently, being covered with enlarged cells containing numerous synthetic organelles or by dividing endothelium (Fig. 3). Often the media appeared damaged as a result of the disruption of the IEL with evidence of smooth muscle cell necrosis and loss (Fig. 3). In these regions leukocytes were observed (Fig. 2) as were smooth muscle cells containing few myofilaments but large numbers of organelles such as rough ER (Figs. 2,6) (synthetic state smooth muscle, see Campbell and Campbell, 1985). In 3 of the 64 SHR renal arteries examined at the bifurcation near the kidney hilus, aneurysms were observed. None were observed in normotensive rats. The significant feature of each of these aneurysms was disruption of the IEL in regions adjacent to the aneurysm itself (Figs. 4-6).
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FIG. 1. Spontaneous lesion in the SHR caudal artery. Note the abrupt breaks in the IEL and the elastic recoil of the IEL at the break points. x 200.
There appeared to be more spontaneous lesions present in arteries of SHR than in those of their normotensive counterparts. However, this observation was not quantitated. Part 2: Experimental
Model
In each case the type of injury and subsequent healing process was morphologically indistinguishable in both the rabbit and the rat, therefore they will be considered together. There was also no difference between the response in male and female rats examined at 3 weeks. Type of injury produced. Two types of circumferential lesion were produced by the method described, a large and a small lesion (Figs. 7- 11,13). The larger lesion was up to 1 mm in length (Figs. 9,lO) and was characterized by the loss of endothelium, rupture of the internal elastic lamellae (IEL), and the rupture of a variable number of medial elastic lamellae. In the largest most severe lesions, all the medial lamellae were ruptured right through to the adventitia (Fig. 10). These large lesions were the ones that became visible during the stretching process. In no cases did any of these produce aneurysms. Within the media itself there appeared to be little, if any, damage to the smooth muscle cells themselves although the extracellular matrix was often disrupted and displaced. Some blood was seen to penetrate the extracellular space in the most severe lesions. Small lesions were up to 400 pm in length (Figs. 7,8,11) and were characterized by the rupture and loss of a small area of endothelium and rupture of the IEL. There was no morphologically detectable damage to the media. Endothelial regeneration. Immediately following injury the subendothelium or exposed media became covered with a platelet carpet and pronounced mi-
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FIG. 2. Part of a spontaneous lesion in a SHR mesenteric artery. Note the abrupt break in the IEL (arrow), the mononuclear cell (M) just below the endothelial surface and the “synthetic state” smooth muscle cells (S). x 5500. FIG. 3. Part of a spontaneous lesion in a SHR mesenteric artery which has recently occurred demonstrating a dividing endothelial cell (e) and evidence of smooth muscle necrosis and loss from the media. x 4000.
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FIG. 4. (A-D) Sequential transverse sections through the SHR renal artery at the level of the bifurcation into smaller renal branches. As the sections are cut closer to the kidney the aneurysm (a) passes out of the plane of section. x 49.
crothrombi in some areas (Fig. 9). This carpet persisted for approximately 4-5 hr. After this time leukocytes were present on the exposed area (Figs. 11,12). The lesions themselves were rapidly recovered with endothelium. After 3.5 hr in the rabbit the endothelium had extended over the angular broken face of the IEL and down onto the exposed media (Figs. 12,15A). The small lesions were
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FIG. 5. Higher magnification of central region of SHR renal artery branch shown in Fig. 4B. Note the discontinuity of the IEL (arrow) and the aneurysm (a). x 400. FIG. 6. Electron micrograph of SHR renal artery branch shown in Figs. 4 and 5 at the level of a spontaneous lesion. Note the abrupt break in the internal elastic lamella (arrow) and the synthetic state smooth muscle cell (S) immediately beneath the surface. x 2000.
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FIG. 7. Small circumferential lesion produced in the rat carotid artery 3 days after injury. Only the intima. IEL, and first lamellar unit have been damaged. Note the similarity of this lesion to the spontaneous lesion shown in Fig. 1. x 100. FIG. 8. Reendothelialized small circumferential lesion from a rat carotid artery 3 days after injury. Note disruption of the IEL and the presence of fibrin beneath the endothelium. x 500.
completely covered with endothelium by 24 hr postinjury (Fig. 13) and at this point the endothelial cells were indistinguishable from cells in uninjured areas. The larger lesions were completely covered by endothelium at 7-10 days, depending on size. By 2 weeks the area was completely covered by elongated endothelial cells resembling those of the surrounding uninjured endothelium.
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FIG. 9. Large circumferential lesion produced in the rabbit carotid artery 3.5 hr after injury showing loss of endothelium over the lesion and rupture of the IEL and a variable number of medial elastic lamellae. Note the pronounced microthrombus on the surface. x 250. FIG. IO. Large circumferential lesion produced in the rabbit carotid artery 3 weeks after injury. In this case all the medial lamellar units were ruptured. Note the involvement of the adventitia in the repair process. The adventitia has become thickened with a marked increase in cellularity. The surface is completely reendothelialized. x 100.
Response of the artery wall. Over the time course studied there was a progressive repair of the artery wall. The normal medial smooth muscle cells of both the rats and rabbits used in the experimental study had a relatively high content of synthetic organelles. During the course of repair there was no readily apparent change in the phenotypic status of any of the cells. In some of the rats at 3 days, a few smooth muscle cells immediately beneath the lumenal surface of the larger lesions appeared to have a slightly higher organelle content. The relatively high organelle content of the cells at all stages may simply reflect the fact that the animals were young and therefore the vasculature was still in the process of active growth and remodelling.
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FIG. 11. Small incomplete circumferential lesion produced in the wall of the rabbit carotid artery 3.5 hr after injury. Note the endothelium overlying the longitudinal corrugations in the IEL. Several leukocytes adhere to the subendothelial connective tissue. x 280.
In both large and small lesions the number of smooth muscle cells immediately beneath the lesion surface appeared to increase slightly. Whether this was due to migration or division is not clear, since few mitotic figures were observed at any stage. In the small lesions which were reendothelialised by 24 hr, the cells immediately beneath the lumenal surface synthesized elastin such that by 3-5 weeks after injury a 3-dimensional elastin lattice work resulted. Beyond this time there was no further change in the lesion. In the larger lesions, repair involved a partial reorganization of previously disrupted lamellar units and the synthesis of collagen and elastin (Fig. 15B). In the largest lesions the adventitia became involved in the response resulting in a thickening and increased cellularity (Fig. 10). The origin of smooth muscle cells in the adventita was not investigated. As with the small lesions over a period of 3-5 weeks a 3-dimensional elastin network was established (Figs. 15B,16). Beyond this time (up to 10 weeks in rabbits and 20 weeks in rats), there was no further response of the wall. In neither the rat nor the rabbit was there at any time an intimal proliferation of smooth muscle cells associated with the lesions. In regions next to large lesions in both rats and rabbits an occasional single layer of longitudinally oriented intimal
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FIG. 12. Part of a large circumferential lesion 24 hr after injury showing the leading edge of endothelium growing in over the exposed subendothelium. The edge of the disrupted IEL can be seen at the far right of the photograph (arrows). Note the leukocytes adhering to the subendothelium. x 1120.
smooth muscle cells was present at around 3-5 days (Figs. 14A,B). This “neointima” did not extend any significant distance from the lesion nor did it undergo any further change over the subsequent weeks. DISCUSSION Disintegration or fragmentation of elastic lamellae of arteries is a well documented phenomenon occurring under both normal conditions such as growth and aging, and pathological conditions such as atherosclerosis, aneurysms, and hypertension and is reviewed below. With growth, the radius and length of arteries increases considerably. This growth is not due solely to a numerical increase in fibers, lamellae, and smooth muscle cells, for in many arteries a profound remodelling occurs after birth resulting in the formation of additional structures. This remodelling occurs across the wall but is most conspicuous in the innermost layer of the growing artery where secondary elastic lamellae form between the endothelium and the primary internal elastic membrane. The development of this layer is particularly marked in human coronary arteries where it has been shown to represent a true aging process (see Winter er al., 1979). At birth the subendothelial layer is thin with few elements present. Within the first few years a second longitudinal “elastic-hyperplastic layer” consisting of concentrically arranged elastic fibers, lamellae, and a
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FIG. 13. Small incomplete circumferential lesion which has been completely reendothelialized 24 hr after injury. The cells are apparently the same size as the ones from the surrounding uninjured area. The abrupt edges of the IEL are visible through the endothelium. x 560.
few smooth muscle cells develops. With advancing age this layer further increases in size to eventually form the so-called “diffuse or fibromuscular intimal thickening” of the adult which can be up to twice the thickness of the coronary artery media (Meyer et al., 1980). Why a significant amount of growth in size of arteries occurs by this process of remodelling is unknown. However, the fact that the elastic lamellae are fenestrated but otherwise continuous sheets or tubes of elastin could be significant. How do these elastic lamellae increase in length? Observations on the formation of intimal thickenings have lead to two schools of thought. One group maintains that there is a progressive splitting or fragmentation of the internal elastic lamina with the remaining lamellae forming a matrix from which the secondary elastic sheets arise (Hartman, 1977). The second group suggests that there is no splitting or fragmentation but merely a stretching with a consequent increase in size of the fenestrations and decrease in thickness of the lamellae (Nemetschek-Gansler et al., 1979). Arteries in aging animals exhibit three prominent morphological features: (i) elastic fragmentation, characterized by disruption of elastic lamellae; (ii) degeneration of smooth muscle cells; and (iii) a concomitant increase in collagen and ground substance, leading to what is termed fibrosis (Schlatmann and Becker, 1977). Similar changes have also been observed in arteries of hypertensive animals (Gardner and Matthews, 1969; Aikawa and Koletsky, 1970).
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FIG. 14. Part of a large lesion 5 days after injury in the rabbit carotid artery. (A) Endothelium extends over the ruptured end of the IEL and over the injured area. Adjacent to the wound, a single neointimal smooth muscle cell can be seen between the endothelium and IEL (arrow). x250. lB) Electron micrograph of the small neointima formed immediately adjacent to a large circumferential lesion. Note the longitudinal orientation of the smooth muscle cell in the neointima. IEL-internal elastic lamella. X 2200.
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FIG. 15. (A, B) Electron micrographs of a larger lesions (A) 3.5 hr and (B) 3 weeks after injury. Disruption of the media is apparent in (A). In (B) reorganization of the media and the synthesis of large amounts of elastin can be seen. (A) x 2800. (B) x 3600.
As early as 1904 Marchand claimed that atherosclerosis begins with the destruction of the elastic elements of the arteries. Lansing (1955) suggested that atherosclerosis is not one disease but two, one of which involves a defect in cholesterol metabolism while the other is manifested by the breakdown of the elastic elements of the arteries. Recently, more interest has centered on the role of degradation of elastin in the development of the atherosclerotic plaque, in view of the finding that elastolytic activity in the human aorta increases with increasing exneutrophils, mactent of atheroma (Hornebeck et al., 1978). Polymorphonuclear rophages, platelets, and smooth muscle cells have now all been shown to produce elastolytic enzymes (Hornebeck et al., 1981a) and recent experiments suggest that purified LDL preparations from human sera induce an increase in an intracellular elastase-like protease in aortic smooth muscle cells, while HDL preparations have no effect (Hornebeck et al., 1981b). Haust (1979) has demonstrated differences between the processes of elastogenesis and degeneration of elastic tissue in explants from normal aorta and those from atherosclerotic lesions of rabbits maintained in culture for 10 days. She suggests this may be due to differences in the smooth muscle cells between the normo and hypercholesterolemit animals. In studies of the formation of aneurysms in the human aorta (Schlatmann and Becker, 1977) and small arteries, such as cerebral vessels (Stehbens, 1981), elastic lamellae fragmentation is a characteristic feature. Further evidence demonstrating elastica damage as an initiating event in aneurysm formation is found in studies of Mar-fan’s Syndrome, which is an inherited connective tissue disease of
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FIG. 16. Large circumferential lesion in the rabbit carotid artery 5 weeks after injury showing the complex 3-dimensional elastin network that has been established. The ruptured edge of the original IEL is visible on the right (large arrow). A new IEL can also be observed (small arrow). x 250. FIG. 17. Wall of anterior mesenteric artery of 18-month-old SHR. Note the irregular arrangement of the elastic lamellae. x 500.
humans. One manifestation of this disorder is aortic dissecting aneurysms. Microscopy of the lesions showed elastic degeneration, disorganized bundles of collagenous fibers, and abnormal deposits of proteoglycans (Saruk and Eisenstein, 1977). Similar lesions can also be produced experimentally by feeding a copper(BAPN-the deficient diet to chicks (O’Dell et al., 1961), or @aminopropionitrile toxic factor in lathyrism) to a variety of animals (Paik and Lalich, 1970). Both these factors inhibit the normal formation of collagen and elastin. The present study describes the presence of aneurysms in the renal artery of SHR. Although a cause and effect relationship cannot be positively identified, it is interesting to note that in all three cases these aneurysms appeared to be associated with sites of spontaneously occurring breaks in the IEL. Renovascular hypertension as a result of fibromuscular hyperplasia (FMH) of
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the renal artery has been reported with increasing frequency during the past decade. Arteries demonstrating FMH have areas of medial thickening secondary to hypertrophy of smooth muscle and fibrous elements. Areas adjacent to such regions consist of thinned intima, disruption of IEL, and a markedly thinned media composed predominantly of fibrous tissue. It has been suggested that disruption of the arterial elastica is the primary defect of FHM, and the areas of medial thickening reflect compensatory muscular hypertrophy, leading to occlusive lesions, whereas thinning of the walls lead to aneurysms. For many years this disorder was considered to be primarily restricted to human renal arteries. However, recently this condition has been found in human carotid, vertebral, and limb arteries, as well as in a variety of arteries in turkeys (see Julian, 1980). The results of Osborne-Pellegrin and colleagues (1979, 1980, 1982, 1983) and those reviewed and described in this paper also suggest that fragmentation of IEL is a relatively common feature in rat arteries as well as those of other species. It could be further suggested that under normal circumstances damage to the vessel wall is rapidly repaired, with some structural alterations, such as reduplication of lamellae, but with no long-term detrimental effects. However, if rupture of IEL occurs in an abnormal vessel, such as one in which there is depletion of smooth muscle due to aging and/or hypertension, the damage cannot be adequately repaired and pathological sequelae such as aneurysm formation may occur. To determine the truth of this statement we have developed a model which morphologically mimics the spontaneous lesions observed in rat caudal and renal arteries. It is hoped this model may not only explain the large number of observations of disruption or fragmentation of IEL in normal conditions but in pathological conditions as well. In 1931 Ssolowjew found that manipulation and longitudinal stretching of rabbit carotid arteries resulted in disruption of elastic lamellae and endothelial discontinuity over that region. When these animals were fed a cholesterol enriched diet, lipid droplets accumulated in smooth muscle cells neighboring the lesion. It is interesting to note that Osborne-Pellegrin and Weill (1983) report that in hypercholesterolaemic rats spontaneous defects in the caudal and renal arteries represent preferential sites for the accumulation of lipids. We have used a modification of Ssolowjew’s technique as a model for these spontaneous lesions. Two types of circumferential lesions can be produced, one relatively large, the other small. These lesions are morphologically identical to those previously described as occurring spontaneously (Osborne-Pellegrin, 1979; Campbell and Campbell, 1980) with small lesions being completely reendothelialized by 24 hr postinjury and large ones by 7-10 days. Only in large lesions were a few smooth muscle cells observed to have migrated and/or proliferated in the intima, there being no production of a neointima of smooth muscle cells similar to that observed when large areas of endothelium are removed with a balloon catheter (see Stemerman and Ross, 1972). In 1976 Clowes et al. reported that if a segment of endothelium less than 1 cm long was removed from the rat carotid artery a neointima of longitudinally oriented smooth muscle cells did not occur in response to this injury. More recently these studies have been verified by Schwartz and co-workers (see Schwartz, 1982; Schwartz et al., 1982). If small tracks (2-20 cells wide) of endothelium are removed, no proliferation of smooth muscle occurs before restitution of a complete endothelial covering (Hirsch and Robertson, 1977; Reidy and Schwartz,
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1981). These experiments demonstrate that there is a critical lesion size, or a time for endothelial regeneration to occur, before smooth muscle cells can proliferate in the intima. It has been suggested that this phenomenon may be related to the fact that in the majority of cases before a smooth muscle cell can proliferate it must modulate its phenotype from a contractile state (a state in which most smooth muscle cells in the artery media exist) to the synthetic state, a process which is time related (see Campbell and Campbell, 1985). However, Reidy and Silver (1985) have recently compared the repair of areas of arterial wall denuded of tracts of endothelium lo-15 cells wide and 90-120 cells wide with time. The smaller injury was reendothelialized by 72 hr and the larger one by 7-8 days. No significant increase in smooth muscle replication was detected in the aortas in either of these injuries. The authors suggest this was due to the fact that the nylon catheter used to remove the endothelium did not inflict any trauma on the wall itself. They hypothesize that medial injury and, in particular, damage to smooth muscle cells is a prerequisite for intimal smooth muscle proliferation. The results of the present paper in which there was extensive damage to the media but few smooth muscle cells damaged and no intimal proliferation, are in agreement with this hypothesis. ACKNOWLEDGMENTS The authors thank Miss Fabian Bowers for typing the manuscript and Miss Violet Cullinan for technical assistance.
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OSBORNE-PELLEGRIN, M. J., and WEILL. D. (1983). “Spontaneous” endothelial injury in the rat caudal artery. Exp. MoZ. Parho/. 39, 61-79. OSBORNE-PELLEGRIN, M. J., WEILL, D.. and COUTARD, M. (1980). A study of the influence of steroid hormones, hemodynamics, and environmental factors on the formation of small “spontaneous” lesions in the rat caudal artery. Lub. Zmaest. 43, 471-479. PAIK, W. C. W., and LALICH, J. J. (1970). Factors which contribute to aortic fibrous repair in rats fed S-aminopropionitrile. Lab. Invest. 22, 28-35. REIDY, M.A., and SCHWARTZ, S. M. (1981). Endothelial regeneration: III. Time course of intimal changes after small defined injury to rat aortic endothelium. Lab. Znvesf. 44, 301-308. REIDY. M. A., and SILVER, M. (1985). Endothelial regeneration. VII. Lack of intimal proliferation after defined injury to rat aorta. Amer. J. Putho/. 118, 173-177. SARUK, M., and EISENSTEIN, R. (1977). Aortic lesion in Marfan syndrome. Arch. Putho/. Lub. Med. 101, 74-77.
SCHLATMANN, T. J. M., and BECKER, A. E. (1977). Histologic changes in the normal aging aorta: Implications for dissecting aortic aneurysm. Amer. J. Cardiol. 39, 13-26. SCHWARTZ, S. M. (1982). Vascular integrity. In “Biologic and Synthetic Vascular Prosthesis” (J. C. Stanley, ed.), pp. 27-36. Grune & Stratton, New York. SCHWARTZ, S. M., GAJDUSEK. C. M., and OWENS, G. K. (1982). Vessel wall growth control. In “Pathobiology of the Endothelial Cell” (H. L. Nossel and H. J. Vogel, eds.), pp. 63-78. Academic Press, New York. SSOLOWIEW, A. (1931). Uber experimentell hervorgerufene Elasticarisse der Arterien und deren Bedeutung fur die Lipoidablagerung. Virchows Arch. 283, 213-230. STEHBENS, W. E. (1981). Arterial structure at branches and bifurcations with reference to physiological and pathological processes, including aneurysm formation. In “Structure and Function of the Circulation” (C. J. Schwartz, N. T. Werthesson, and S. Wolf, eds.), Vol. II, pp. 667-694. Plenum, New York. STEMERMAN, M. B., and Ross, R. (1972). Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study. .Z.Exp. Med. 136, 769-789. WEXLER, B. C. (1964). Spontaneous arteriosclerosis in repeatedly bred male and female rats. .Z.Atheroscler.
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WEXLER, B. C. (1970). Arteriosclerosis Atherosclerosis
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WINTER, M., HELLETZGRUBER, M., OPPOLZER, R., and SINZINGER, H. (1979). Alterations internal elastic membrane in juvenile human arteries. Actu Anut. 105, 524-528.
in the
AND
MOLECULAR
Spontaneous
PATHOLOGY
45, 227-244 (1986)
Arterial Lesions: Their in Arteriosclerosis
Possible
Role
GORDON R. CAMPBELL AND PETER R. L. MOSSE Department
of Anatomy, Received
University
Noijember
Il.
of Melbourne.
Parkville,
1985, and in revisedform
3052,
Victoria.
April
22. 1986
Australia
Spontaneous lesions of the arterial wall involving the internal elastic lamellae and variable amounts of the intima are described in the spontaneously hypertensive rat caudal. renal, and mesenteric arteries. A simple model for producing similar circumferential lesions in rat and rabbit carotid arteries has been developed and the subsequent repair of these lesions is described. Two types of circumferential lesion can be produced by the application of 50- 160 g of longitudinally applied tension. Small lesions can be up to 400 km in length and are characterized by the loss of a small area of endothelium and rupture of the internal elastic lamellae. No demonstrable damage to the media is detected in these lesions. Larger lesions can be up to 1 mm in length and are characterized by the loss of endothelium and rupture of the internal as well as a variable number of medial elastic lamellae. Little, if any, damage to the medial smooth muscle ceils is observed although the extracellular matrix is often disrupted. Small lesions are completely reendothelialized within 24 hr and larger lesions within 7-10 days. Both large and small lesions repair without the formation of an intimal thickening of smooth muscle cells, despite quite marked damage to the media of the larger lesions. 0 1986 Academic Press. Inc.
INTRODUCTION The major components of the aortic media are smooth muscle cells, elastic lamellae, collagen, and proteoglycans, which together form complex, integrated functional units (Glagov, 1979). Throughout the vascular system the contribution of each of these components with its differing properties varies according to the functional demands placed upon the particular vessel. The relationship between components is dynamic in nature with the unit capable of remodelling its structure to accommodate any change in demands, as for instance in growth and hypertension (see Campbell and Charnley-Campbell, 1981). Changes within one component of the vessel wall, whether normal or pathological, affects others. One function of the elastic lamellae of arteries is to distribute the tensile stresses uniformly throughout the wall (Glagov, 1979). Any dramatic change in these lamellae, such as fragmentation or loss of structural integrity leads to a localized concentration of stress at this site. Depending on the size of the lesion and condition of the vessel wall at that point, a sudden increase in pressure may in turn lead to pathological consequences. In 1979 Osborne-Pellegrin reported “spontaneous” lesions in the caudal artery of mature male Wistar rats. These lesions consisted of regions where the internal elastic lamellae (IEL) was absent over all or part of the vessel circumference. In some cases after rupture the lamellae together with the overlying endothelial cells had curled back along the vessel. Further studies by the same authors suggested flow rate and growth were important factors in the formation of these lesions (Osborne-Pellegrin ef al., 1980; Osborne-Pellegrin and Weill, 1983). In 1980 we reported a similar phenomenon in rat renal arteries (Campbell and Campbell, 227 0014-4800/86 $3.00 Copyright k5 1986 hy Academic Press, Inc. All rIghI* of reproduction in any form reserved.
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1980) and these observations have been later contirmed by Osborne-Pellegrin and co-workers (Coutard and Osborne-Pellegrin, 1982). The present article describes “spontaneous” lesions and their apparent consequences in the renal and mesenteric arteries of old hypertensive rats. A model is also described for producing similar lesions in rat and rabbit carotid artery, and the repair mechanisms following such damage examined. These data are compared with reports of repair to “spontaneous” injury (Osborne-Pellegrin and Weill, 1983). MATERIALS Part 1: Spontaneous
AND METHODS
Lesions
Caudal, renal, and mesenteric arteries of 4- to 18-month-old Kyoto-Wistar spontaneously hypertensive (SHR) (blood pressures > 180 mm Hg), and normotensive male rats were examined in this study. The renal artery was examined in two regions, just proximal to the suprarenal branch and at the bifurcation into the smaller renal arteries, close to the hilus of the kidney. A variety of mesenteric arteries of differing sizes was chosen for examination. Sixty-three SHR and 46 normotensive animals were used. The rats were anesthetized with ether, their heart exposed, and a cannula inserted into the left ventricle. The left jugular vein was exposed and cut at the commencement of perfusion. After initial perfusion washout with 0.1 It4 phosphate buffer, pH 7.3, the animal was fixed with 5% glutaraldehyde in 0.1 M phosphate buffer at pressure approximating that in situ for 15 min. The above arteries were quickly dissected free and further fixed for 1 hr, washed overnight in buffer, and postfixed in 1% osmium tetroxide for 1 hr. Part 2: Experimental Model Sixteen male New Zealand white rabbits (1.5-2.5 kg) and 21 female and 3 male Sprague-Dawley rats (200-250 g) were used. Rabbits were maintained on a daily ration of 100 g of pellet food with greens once a week. Rats were allowed free access to pellet food. Experimental procedure. Prior to surgery rats were anesthetized with ether; rabbits were preanesthetized with Epontal (Bayer Australia), intubated and maintained on Halothane (ICI Australia). In both rats and rabbits the left and right common carotid arteries were exposed by a mid-ventral incision in the neck. The arteries were cleared of surrounding connective tissue and nerves and two double loop ties were passed around the artery. These ties were separated by a distance of approximately 1 cm in rats and 1 to 1.5 cm in rabbits. Longitudinal tension was applied via these loops and vessel wall integrity monitored through x 2 operating glasses. At the first sign of damage, tension was released and the ties quickly removed. The amount of tension required to produce damage varied between animals, therefore no attempt was made to use a uniform tension each time. Between 50 and 160 g tension was required to produce the lesion in both rats and rabbits. The animals were closed and the rabbits were given an intramuscular injection of the antibiotic Reverin (Rolitetracycline 50 mg/kg, Hoechst). Fixation. Two rabbits were examined at each of 3.5-hr, l-day, 3-day, 5-day, 7-day, IO-day, 5-week, and IO-week time intervals. Three rats were examined at each of 2-hr, 3-day, 3-week, 6-week, 12-week, and 20-week intervals. At the 3week time interval, three females and three males were examined for comparison.
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Prior to perfusion fixation rats were anesthetized with ether and rabbits with intravenous sodium pentobarbitol. The abdominal aorta was exposed and a retrograde cannula introduced. The left jugular vein was exposed and cut at the start of perfusion. The animals were perfused with Hanks’ balanced salt solution (CSL, Melbourne, Australia) until the perfusate from the vein was clear of blood. The animal was then fixed with 5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, at high pressure for 5- 10 min. Both carotids were quickly removed and immersed in fixative for a further 1 hr, washed in 0.1 M phosphate buffer, pH 7.3, and postfixed in 1% osmium tetroxide for 1 hr. Only areas between the two ties were examined since in some cases the ties caused trauma to the vessel wall. Tissue for scanning electron microscopy was subsequently dehydrated in a graded ethanol series, critical point dried with carbon dioxide, and gold coated in an Edwards sputter coater. Specimens were viewed in an Etec Autoscan scanning electron microscope at 30 kV. Light and transmission electron microscopy. Tissue for light and transmission electron microscopy was dehydrated in a graded acetone series and embedded in Epon/Araldite. Thick sections were cut on glass knives and stained with methylene blue. Thin sections were cut on diamond knives, stained with uranyl acetate and lead citrate and viewed either in a Siemens or a Philips EM400 electron microscope at 60 kV. RESULTS Part I: Spontaneous
Lesions
Spontaneous lesions in rat caudal and renal arteries have been described in detail in previous reports (Wexler, 1964, 1970; Osborne-Pellegrin, 1979; Campbell and Campbell, 1980; Osborne-Pellegrin and Weill, 1983). Therefore only a brief description will be provided for comparison with the experimental lesions described in Part 2. A typical lesion in the SHR caudal artery is shown in Fig. 1. Here, the IEL is absent over a region of approximately 100 km and folded back at the edges as if it has snapped under tension. The area between the edges is reendothelialized and the media has in this case been little affected by the lamellar changes. Lesions similar to those described in renal and caudal arteries were also observed in a range of different sized mesenteric arteries. Since these lesions also appear to develop randomly, different stages of repair could be observed in different regions, even of the same artery. A number of lesions appeared to have been reendothelialized some time previously as they were covered with apparently normal endothelium (Figs. 2,6). However, the most common lesions were those which appeared to have been reendothelialized recently, being covered with enlarged cells containing numerous synthetic organelles or by dividing endothelium (Fig. 3). Often the media appeared damaged as a result of the disruption of the IEL with evidence of smooth muscle cell necrosis and loss (Fig. 3). In these regions leukocytes were observed (Fig. 2) as were smooth muscle cells containing few myofilaments but large numbers of organelles such as rough ER (Figs. 2,6) (synthetic state smooth muscle, see Campbell and Campbell, 1985). In 3 of the 64 SHR renal arteries examined at the bifurcation near the kidney hilus, aneurysms were observed. None were observed in normotensive rats. The significant feature of each of these aneurysms was disruption of the IEL in regions adjacent to the aneurysm itself (Figs. 4-6).
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FIG. 1. Spontaneous lesion in the SHR caudal artery. Note the abrupt breaks in the IEL and the elastic recoil of the IEL at the break points. x 200.
There appeared to be more spontaneous lesions present in arteries of SHR than in those of their normotensive counterparts. However, this observation was not quantitated. Part 2: Experimental
Model
In each case the type of injury and subsequent healing process was morphologically indistinguishable in both the rabbit and the rat, therefore they will be considered together. There was also no difference between the response in male and female rats examined at 3 weeks. Type of injury produced. Two types of circumferential lesion were produced by the method described, a large and a small lesion (Figs. 7- 11,13). The larger lesion was up to 1 mm in length (Figs. 9,lO) and was characterized by the loss of endothelium, rupture of the internal elastic lamellae (IEL), and the rupture of a variable number of medial elastic lamellae. In the largest most severe lesions, all the medial lamellae were ruptured right through to the adventitia (Fig. 10). These large lesions were the ones that became visible during the stretching process. In no cases did any of these produce aneurysms. Within the media itself there appeared to be little, if any, damage to the smooth muscle cells themselves although the extracellular matrix was often disrupted and displaced. Some blood was seen to penetrate the extracellular space in the most severe lesions. Small lesions were up to 400 pm in length (Figs. 7,8,11) and were characterized by the rupture and loss of a small area of endothelium and rupture of the IEL. There was no morphologically detectable damage to the media. Endothelial regeneration. Immediately following injury the subendothelium or exposed media became covered with a platelet carpet and pronounced mi-
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FIG. 2. Part of a spontaneous lesion in a SHR mesenteric artery. Note the abrupt break in the IEL (arrow), the mononuclear cell (M) just below the endothelial surface and the “synthetic state” smooth muscle cells (S). x 5500. FIG. 3. Part of a spontaneous lesion in a SHR mesenteric artery which has recently occurred demonstrating a dividing endothelial cell (e) and evidence of smooth muscle necrosis and loss from the media. x 4000.
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FIG. 4. (A-D) Sequential transverse sections through the SHR renal artery at the level of the bifurcation into smaller renal branches. As the sections are cut closer to the kidney the aneurysm (a) passes out of the plane of section. x 49.
crothrombi in some areas (Fig. 9). This carpet persisted for approximately 4-5 hr. After this time leukocytes were present on the exposed area (Figs. 11,12). The lesions themselves were rapidly recovered with endothelium. After 3.5 hr in the rabbit the endothelium had extended over the angular broken face of the IEL and down onto the exposed media (Figs. 12,15A). The small lesions were
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FIG. 5. Higher magnification of central region of SHR renal artery branch shown in Fig. 4B. Note the discontinuity of the IEL (arrow) and the aneurysm (a). x 400. FIG. 6. Electron micrograph of SHR renal artery branch shown in Figs. 4 and 5 at the level of a spontaneous lesion. Note the abrupt break in the internal elastic lamella (arrow) and the synthetic state smooth muscle cell (S) immediately beneath the surface. x 2000.
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FIG. 7. Small circumferential lesion produced in the rat carotid artery 3 days after injury. Only the intima. IEL, and first lamellar unit have been damaged. Note the similarity of this lesion to the spontaneous lesion shown in Fig. 1. x 100. FIG. 8. Reendothelialized small circumferential lesion from a rat carotid artery 3 days after injury. Note disruption of the IEL and the presence of fibrin beneath the endothelium. x 500.
completely covered with endothelium by 24 hr postinjury (Fig. 13) and at this point the endothelial cells were indistinguishable from cells in uninjured areas. The larger lesions were completely covered by endothelium at 7-10 days, depending on size. By 2 weeks the area was completely covered by elongated endothelial cells resembling those of the surrounding uninjured endothelium.
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FIG. 9. Large circumferential lesion produced in the rabbit carotid artery 3.5 hr after injury showing loss of endothelium over the lesion and rupture of the IEL and a variable number of medial elastic lamellae. Note the pronounced microthrombus on the surface. x 250. FIG. IO. Large circumferential lesion produced in the rabbit carotid artery 3 weeks after injury. In this case all the medial lamellar units were ruptured. Note the involvement of the adventitia in the repair process. The adventitia has become thickened with a marked increase in cellularity. The surface is completely reendothelialized. x 100.
Response of the artery wall. Over the time course studied there was a progressive repair of the artery wall. The normal medial smooth muscle cells of both the rats and rabbits used in the experimental study had a relatively high content of synthetic organelles. During the course of repair there was no readily apparent change in the phenotypic status of any of the cells. In some of the rats at 3 days, a few smooth muscle cells immediately beneath the lumenal surface of the larger lesions appeared to have a slightly higher organelle content. The relatively high organelle content of the cells at all stages may simply reflect the fact that the animals were young and therefore the vasculature was still in the process of active growth and remodelling.
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FIG. 11. Small incomplete circumferential lesion produced in the wall of the rabbit carotid artery 3.5 hr after injury. Note the endothelium overlying the longitudinal corrugations in the IEL. Several leukocytes adhere to the subendothelial connective tissue. x 280.
In both large and small lesions the number of smooth muscle cells immediately beneath the lesion surface appeared to increase slightly. Whether this was due to migration or division is not clear, since few mitotic figures were observed at any stage. In the small lesions which were reendothelialised by 24 hr, the cells immediately beneath the lumenal surface synthesized elastin such that by 3-5 weeks after injury a 3-dimensional elastin lattice work resulted. Beyond this time there was no further change in the lesion. In the larger lesions, repair involved a partial reorganization of previously disrupted lamellar units and the synthesis of collagen and elastin (Fig. 15B). In the largest lesions the adventitia became involved in the response resulting in a thickening and increased cellularity (Fig. 10). The origin of smooth muscle cells in the adventita was not investigated. As with the small lesions over a period of 3-5 weeks a 3-dimensional elastin network was established (Figs. 15B,16). Beyond this time (up to 10 weeks in rabbits and 20 weeks in rats), there was no further response of the wall. In neither the rat nor the rabbit was there at any time an intimal proliferation of smooth muscle cells associated with the lesions. In regions next to large lesions in both rats and rabbits an occasional single layer of longitudinally oriented intimal
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FIG. 12. Part of a large circumferential lesion 24 hr after injury showing the leading edge of endothelium growing in over the exposed subendothelium. The edge of the disrupted IEL can be seen at the far right of the photograph (arrows). Note the leukocytes adhering to the subendothelium. x 1120.
smooth muscle cells was present at around 3-5 days (Figs. 14A,B). This “neointima” did not extend any significant distance from the lesion nor did it undergo any further change over the subsequent weeks. DISCUSSION Disintegration or fragmentation of elastic lamellae of arteries is a well documented phenomenon occurring under both normal conditions such as growth and aging, and pathological conditions such as atherosclerosis, aneurysms, and hypertension and is reviewed below. With growth, the radius and length of arteries increases considerably. This growth is not due solely to a numerical increase in fibers, lamellae, and smooth muscle cells, for in many arteries a profound remodelling occurs after birth resulting in the formation of additional structures. This remodelling occurs across the wall but is most conspicuous in the innermost layer of the growing artery where secondary elastic lamellae form between the endothelium and the primary internal elastic membrane. The development of this layer is particularly marked in human coronary arteries where it has been shown to represent a true aging process (see Winter er al., 1979). At birth the subendothelial layer is thin with few elements present. Within the first few years a second longitudinal “elastic-hyperplastic layer” consisting of concentrically arranged elastic fibers, lamellae, and a
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FIG. 13. Small incomplete circumferential lesion which has been completely reendothelialized 24 hr after injury. The cells are apparently the same size as the ones from the surrounding uninjured area. The abrupt edges of the IEL are visible through the endothelium. x 560.
few smooth muscle cells develops. With advancing age this layer further increases in size to eventually form the so-called “diffuse or fibromuscular intimal thickening” of the adult which can be up to twice the thickness of the coronary artery media (Meyer et al., 1980). Why a significant amount of growth in size of arteries occurs by this process of remodelling is unknown. However, the fact that the elastic lamellae are fenestrated but otherwise continuous sheets or tubes of elastin could be significant. How do these elastic lamellae increase in length? Observations on the formation of intimal thickenings have lead to two schools of thought. One group maintains that there is a progressive splitting or fragmentation of the internal elastic lamina with the remaining lamellae forming a matrix from which the secondary elastic sheets arise (Hartman, 1977). The second group suggests that there is no splitting or fragmentation but merely a stretching with a consequent increase in size of the fenestrations and decrease in thickness of the lamellae (Nemetschek-Gansler et al., 1979). Arteries in aging animals exhibit three prominent morphological features: (i) elastic fragmentation, characterized by disruption of elastic lamellae; (ii) degeneration of smooth muscle cells; and (iii) a concomitant increase in collagen and ground substance, leading to what is termed fibrosis (Schlatmann and Becker, 1977). Similar changes have also been observed in arteries of hypertensive animals (Gardner and Matthews, 1969; Aikawa and Koletsky, 1970).
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FIG. 14. Part of a large lesion 5 days after injury in the rabbit carotid artery. (A) Endothelium extends over the ruptured end of the IEL and over the injured area. Adjacent to the wound, a single neointimal smooth muscle cell can be seen between the endothelium and IEL (arrow). x250. lB) Electron micrograph of the small neointima formed immediately adjacent to a large circumferential lesion. Note the longitudinal orientation of the smooth muscle cell in the neointima. IEL-internal elastic lamella. X 2200.
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FIG. 15. (A, B) Electron micrographs of a larger lesions (A) 3.5 hr and (B) 3 weeks after injury. Disruption of the media is apparent in (A). In (B) reorganization of the media and the synthesis of large amounts of elastin can be seen. (A) x 2800. (B) x 3600.
As early as 1904 Marchand claimed that atherosclerosis begins with the destruction of the elastic elements of the arteries. Lansing (1955) suggested that atherosclerosis is not one disease but two, one of which involves a defect in cholesterol metabolism while the other is manifested by the breakdown of the elastic elements of the arteries. Recently, more interest has centered on the role of degradation of elastin in the development of the atherosclerotic plaque, in view of the finding that elastolytic activity in the human aorta increases with increasing exneutrophils, mactent of atheroma (Hornebeck et al., 1978). Polymorphonuclear rophages, platelets, and smooth muscle cells have now all been shown to produce elastolytic enzymes (Hornebeck et al., 1981a) and recent experiments suggest that purified LDL preparations from human sera induce an increase in an intracellular elastase-like protease in aortic smooth muscle cells, while HDL preparations have no effect (Hornebeck et al., 1981b). Haust (1979) has demonstrated differences between the processes of elastogenesis and degeneration of elastic tissue in explants from normal aorta and those from atherosclerotic lesions of rabbits maintained in culture for 10 days. She suggests this may be due to differences in the smooth muscle cells between the normo and hypercholesterolemit animals. In studies of the formation of aneurysms in the human aorta (Schlatmann and Becker, 1977) and small arteries, such as cerebral vessels (Stehbens, 1981), elastic lamellae fragmentation is a characteristic feature. Further evidence demonstrating elastica damage as an initiating event in aneurysm formation is found in studies of Mar-fan’s Syndrome, which is an inherited connective tissue disease of
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FIG. 16. Large circumferential lesion in the rabbit carotid artery 5 weeks after injury showing the complex 3-dimensional elastin network that has been established. The ruptured edge of the original IEL is visible on the right (large arrow). A new IEL can also be observed (small arrow). x 250. FIG. 17. Wall of anterior mesenteric artery of 18-month-old SHR. Note the irregular arrangement of the elastic lamellae. x 500.
humans. One manifestation of this disorder is aortic dissecting aneurysms. Microscopy of the lesions showed elastic degeneration, disorganized bundles of collagenous fibers, and abnormal deposits of proteoglycans (Saruk and Eisenstein, 1977). Similar lesions can also be produced experimentally by feeding a copper(BAPN-the deficient diet to chicks (O’Dell et al., 1961), or @aminopropionitrile toxic factor in lathyrism) to a variety of animals (Paik and Lalich, 1970). Both these factors inhibit the normal formation of collagen and elastin. The present study describes the presence of aneurysms in the renal artery of SHR. Although a cause and effect relationship cannot be positively identified, it is interesting to note that in all three cases these aneurysms appeared to be associated with sites of spontaneously occurring breaks in the IEL. Renovascular hypertension as a result of fibromuscular hyperplasia (FMH) of
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the renal artery has been reported with increasing frequency during the past decade. Arteries demonstrating FMH have areas of medial thickening secondary to hypertrophy of smooth muscle and fibrous elements. Areas adjacent to such regions consist of thinned intima, disruption of IEL, and a markedly thinned media composed predominantly of fibrous tissue. It has been suggested that disruption of the arterial elastica is the primary defect of FHM, and the areas of medial thickening reflect compensatory muscular hypertrophy, leading to occlusive lesions, whereas thinning of the walls lead to aneurysms. For many years this disorder was considered to be primarily restricted to human renal arteries. However, recently this condition has been found in human carotid, vertebral, and limb arteries, as well as in a variety of arteries in turkeys (see Julian, 1980). The results of Osborne-Pellegrin and colleagues (1979, 1980, 1982, 1983) and those reviewed and described in this paper also suggest that fragmentation of IEL is a relatively common feature in rat arteries as well as those of other species. It could be further suggested that under normal circumstances damage to the vessel wall is rapidly repaired, with some structural alterations, such as reduplication of lamellae, but with no long-term detrimental effects. However, if rupture of IEL occurs in an abnormal vessel, such as one in which there is depletion of smooth muscle due to aging and/or hypertension, the damage cannot be adequately repaired and pathological sequelae such as aneurysm formation may occur. To determine the truth of this statement we have developed a model which morphologically mimics the spontaneous lesions observed in rat caudal and renal arteries. It is hoped this model may not only explain the large number of observations of disruption or fragmentation of IEL in normal conditions but in pathological conditions as well. In 1931 Ssolowjew found that manipulation and longitudinal stretching of rabbit carotid arteries resulted in disruption of elastic lamellae and endothelial discontinuity over that region. When these animals were fed a cholesterol enriched diet, lipid droplets accumulated in smooth muscle cells neighboring the lesion. It is interesting to note that Osborne-Pellegrin and Weill (1983) report that in hypercholesterolaemic rats spontaneous defects in the caudal and renal arteries represent preferential sites for the accumulation of lipids. We have used a modification of Ssolowjew’s technique as a model for these spontaneous lesions. Two types of circumferential lesions can be produced, one relatively large, the other small. These lesions are morphologically identical to those previously described as occurring spontaneously (Osborne-Pellegrin, 1979; Campbell and Campbell, 1980) with small lesions being completely reendothelialized by 24 hr postinjury and large ones by 7-10 days. Only in large lesions were a few smooth muscle cells observed to have migrated and/or proliferated in the intima, there being no production of a neointima of smooth muscle cells similar to that observed when large areas of endothelium are removed with a balloon catheter (see Stemerman and Ross, 1972). In 1976 Clowes et al. reported that if a segment of endothelium less than 1 cm long was removed from the rat carotid artery a neointima of longitudinally oriented smooth muscle cells did not occur in response to this injury. More recently these studies have been verified by Schwartz and co-workers (see Schwartz, 1982; Schwartz et al., 1982). If small tracks (2-20 cells wide) of endothelium are removed, no proliferation of smooth muscle occurs before restitution of a complete endothelial covering (Hirsch and Robertson, 1977; Reidy and Schwartz,
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1981). These experiments demonstrate that there is a critical lesion size, or a time for endothelial regeneration to occur, before smooth muscle cells can proliferate in the intima. It has been suggested that this phenomenon may be related to the fact that in the majority of cases before a smooth muscle cell can proliferate it must modulate its phenotype from a contractile state (a state in which most smooth muscle cells in the artery media exist) to the synthetic state, a process which is time related (see Campbell and Campbell, 1985). However, Reidy and Silver (1985) have recently compared the repair of areas of arterial wall denuded of tracts of endothelium lo-15 cells wide and 90-120 cells wide with time. The smaller injury was reendothelialized by 72 hr and the larger one by 7-8 days. No significant increase in smooth muscle replication was detected in the aortas in either of these injuries. The authors suggest this was due to the fact that the nylon catheter used to remove the endothelium did not inflict any trauma on the wall itself. They hypothesize that medial injury and, in particular, damage to smooth muscle cells is a prerequisite for intimal smooth muscle proliferation. The results of the present paper in which there was extensive damage to the media but few smooth muscle cells damaged and no intimal proliferation, are in agreement with this hypothesis. ACKNOWLEDGMENTS The authors thank Miss Fabian Bowers for typing the manuscript and Miss Violet Cullinan for technical assistance.
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