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Derivation of intimal cells appearing in spontaneous healing process of hypertensive arterial lesions
EXPERIMENTAL
Ah‘D
Derivation
MOLECULAR
23, 402-416
( 1975)
of Intimal Cells Appearing in Spontaneous Process of Hypertensive Arterial Lesions KEIJI
Second
PATHOLOGY
SUZUKI,
Department
Received
SADAO
Honr,
AND
GENJU
of Pathology, School of Medicine, Maebashi, Japan March
20> 197.5,
and in revised
form
Healing
OONE~A Gunma
May
Univekty,
20, 1975
Light and electron microscopic and autoradiographic studies were made on the process of spontaneous organization of fibrinoid substance deposited in the intima, that is, spontaneous healing of intimal fibrinoid degeneration of the hypertensive rat mesenteric arteries at 8-24 weeks after the constriction of the bilateral renal arteries. The organization of the intimal fibrinoid substance by intimal cells was performed mainly from the luminal side and infrequently from the medial side. Indigenous intimal cells and circulating blood cells were nat positively involved in the organization. During the organization of fibrinoid substance, the endothelial cetls turned into smooth muscle cell-like cells in situ, and there was a picture s~l~~esting the migration of these smooth muscle cell-like endothehal rells into the intima or their incorporation into the intima by the covering of adjacent endothelial cells. Experiments with intravenous administration of W-proline revealed that endothelial cells and intimal and medial smooth muscle cells resembled each other functionally. It can therefore be said that the majority of the cells that participate in the organization of intimal fibrinoid substance are intimal smooth muscle cells derived from endothelial cells, and a part of the cells is derived from medial smooth muscle cells. The cellulofibrous tissue of the intima after the complete organization of intimal fibrinoid substance resulted from the proliferation of the en(~~tIlelial cellderived intimal smooth muscle cells and from the fiber and ground substance formation by these.
INTRODUCTION There are many theories concerning the derivation of smooth muscle cells constituting arterial intimal thickening, and no consensus has yet been attained, Among them are found the following: ( 1) medial smooth muscle cell-derivation theory (Filshie and Scott, f958; Consta~tinides et al, 1958; Buck, 1961; Luginbiihl and Johes, 1965; Murray et al., 1966; Parker and Odland, 1966; Wissler, 1967; Jurukova and Rohr, 1968; Aikawa and Koletsky, 1970); (2) endothelial cellderivation theory (Mehrotra, 1953; Haust et al., 1960; Takeda, 1961; Ooyama, 1962; Suto, 1964; Sekiguchi, 1964; Kojimahara et al., 197&b); (3) circulating blood cell-derivation theory ( O’Neal et al., 1964; Halpert et al., 1966; Still et al., 1967); (4) in t imal cell-derivation theory (Meessen, 1964); and (5) plural cell derivation theory, which asserts that not any single kind but plural kinds of cells, such as medial smooth muscle cells, endothelial cells, proper intimal 402 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any farm reserved.
DERIVATION
OF INTIMAL
CELLS
403
smooth muscle cells, and circulating blood cells, can be the origin of the increased intimal cells, contributing to intimal thickening (Sawatari, 1966; Suzuki, 1967; Borgers et al., 1973; Ooneda et al., 1973a). Fibrinoid substance, deposited in the arterial intima of experimental hypertensive rats, was organized to form cellulofibrous intimal thickening spontaneously (Takatama, 1960; Suzuki et al., 1972) or after removing the constricting clamps from the renal arteries (Se~~chi, 1964) or after giving antihypertensive drugs (Kojimahara, 1967; Kojimahara et al., 1971a,b). The lesions represented a kind of arteriosclerosis. In the present study, spontaneous organizing process of intima1 fibrinoid substance, i.e., spontaneous healing of intimal fibrinoid degeneration, in the mesenteric and coronary arteries of hypertensive rats was investigated at 8-24 weeks after bilateral renal artery constriction by light and electron microscopy and autoradiography, and the derivation of intimal cells, which increased in the intima, was studied. MATERIAL
AND METHODS
Hypertension was experimentally induced in 61 male Wistar rats, weighing 60-80 gm, by constricting the bilateral renaI arteries with silver clamps 0.23 mm in internal diameter. The animals were sacrificed at 842 postoperative weeks, when their systolic blood pressure was above 160 mm Hg and nodular lesions were found in the mesenteric arteries. Light Microscopic Observation Thirty rats were laparotomized under ether anesthesia, and a lower part of the abdominal aorta was cut to kill them by bleeding. After the sacrifice, the mesenteric arteries showing nodular lesions were taken out and fixed with 1070 buffered neutra1 formalin. After the tissues were dehydrated with graded ethanol and embedded in paraffin, thin sections were prepared, which were stained with HE, Mallory, Weigert’s elastic fiber, and Alcian blue stains. Electron Microscopic Observation Twenty rats were laparotomized under ether anesthesia, the mesenteric arteries and abdominal aorta were exposed, and the latter was clamped above and below the arising sites of the former to stop blood flow. Then a 170 osmic acid was injected into this aortic segment to perfuse the mesenteric arteries with it, Thereafter, the mesenteric arteries with nodular lesions were removed and refixed with a 1% osmic acid for 2 hr. After dehydration and embedding with Epon 812, ultrathin sections were prepared and double stained with uranyl acetate and lead hydroxide to be observed electron microscopically. The coronary arteries treated similarly were also examined by electron microscopy in five animals. Light Microscopic Autoradiographic
Observation
Seven hypertensive rats with well-developed mesenteric nodular lesions were injected with 3H-prohne ( L-xH-proline, specific activity: 3.02 Ci/mmole, New England Nuclear Corporation, Boston, Mass. ) at a dose of 5 #&‘gm body
404
SUZUKI,
HORI
AND
OONEDA
weight through the femoral vein. At 5, 15, 30, 60, and 120 min thereafter, a proximal and a distal arterial part of the noduIar lesions were ligated, and then the intervening arterial segments were removed from four animals. Moreover, at 4, 6, and 24 hr after the intravenous injection of 3H-proline, a nodular arterial segment was taken off from each of three animals. After formalin fixation, paraffin sections were prepared and deparaffinized and then coated with Sakura autoradiograph emulsion NR-M2 (Konishiroku Photo Ind. Co., Ltd., Tokyo) to be exposed to irradiation in a shaded box for 2-3 weeks. After development with Konidol X (KoI~ish~oku Photo Ind. CO., Ltd., Tokyo) and fixation with Konifix (Konishiroku Photo Ind. CO., Ltd., Tokyo), the sections were subjected to nuclear staining with hematoxylin and observed light microscopically. Electron Microscopic Autodiographic
Observation
Four animals were injected with “H-proline (10 ~Ci/gm) mto the femoral vein and, at 30 and 120 min thereafter, the nodular arterial lesions were removed. They were fixed with a 1% osmic acid for 2 hr and then dehydrated and embedded with Epon 812. Ultrathin sections from these tissues were coated with Sakura autoradiographic emulsion NR-H2 (Konishiroku Photo Ind. Co., Ltd., Tokyo), exposed to irradiation for l-2 months, developed with Konidol X and fixed with Konifix, and double stained with uranyl acetate and lead hydroxide to be examined electron microscopically.
The nodular arterial lesions under the light microscope proved to be very similar to the plasmatic arterionecrosis ( Ooneda et al., 197310). Early and late lesions were seen in the rat mesenteric arteries at S-16 postoperative weeks. After 16 weeks, most of them were organized. Eady lesions. Numerous neutrophils were attached to the endotheliai surface. The intima was infiltrated with neutrophils, and plasma ~onlponents were accumulated in it. Medial smooth muscle cells became necrotic, and numerous neutrophils and nuclear fragments were seen in the media. In the adventitia there were marked proliferation of fibroblasts and conspicuous neutrophil infiltration. In the arteries with abundant accuInulation of fibrinoid substance in the intima, the attachment of neutrophils to the endothelial surface was not seen, and endothelial cells turned rather flat with basophilic cytoplasm, nuclear chromatin being increased. The fibrinoid substance in the intima was stained red with HE stain and seen as the accumulation of trabecular or rod-shaped substances. Neither the intima nor the media showed marked neutrophil in~ltratio~l. Generally in the arteries showing fibrinoid necrosis the media became atrophic and the medial cells were reduced or even absent, The nuclei of remaining medial cells were enlarged and their cytoplasm became basophilic, but the migration of the medial smooth muscle cells into the intima was not noticed. Organized Lesions.Endothelial cells were enlarged and proliferated, showing rather basophilic cytoplasm and increased nuclear chromatin. In the early stage of the orgal~ization, the staining property of the subendothelial fibrinoid sub-
DERIVATION
OF INTXMAL
CELLS
FIG. 1. Early stage of organization of intimal fibrinoid substance in the mesenteric artery. Intimal cells are seen just beneath the endotheliar cells, which cover the fibrinoid substance (F) deposited in the intima. Migration of the medial cells into the intima is not seen. Necrotic change of the medial smooth muscle cells is conspicuous. Hypertensive rat 12 weeks after bilateral renal artery constriction. M: media. HE stain.
stance was decreased, Intimal cells appeared beneath the endothelium, and the fibrinoid substance around the intimal cells disappeared (Fig. 1). Weigert’s elastic stain revealed newly formed granular and fibrillar elastic fibers just beneath the endothelial cells and around the intimal cells (Fig. 2). A large amount of acid mucopolysaccharides stained with Alcian blue was noticed around the intimaI cells and beneath the endothelial cells.
FIG. 2. In the intima with fibrinoid substance (F), newly formed granular elastin (arrow) is seen directly beneath the endothelial cells. Mesenteric artery of a hypertensive rat 12 weeks after bilateral renal artery constriction. Weigert’s elastic fiber stain.
406
SUZUKI,
HORI
AND
OONEDA
FIG. 3. Organization of intimal fibrinoid .&stance. Intimal cells are increased in the inner intima, while fibrinoid substance (F) remains in the deep intima. The organization from the media (M) is not marked. hlesenteric artery of a hypertensive rat 12 weeks after hilateral renal artery constriction. HE stain. The intimal fibrinoid substance was organized from the luminal side, and intimal cells that proliferated in the subendothelium entered into the fibrinoid substance, thus decreasing its staining property around them. When the organization advanced, intimal cells were markedly proliferated, often presenting mitotic figures, and elastin and ground substance were formed between the cells, fibrinoid substance remaining only in the deep intima (Fig. 3). It was infrequent that the organization of intima1 fibrinoid substance was performed from the medial side. Thus, on rare occasions, intimal fibrinoid substance was penetrated by medial smooth muscle cells, when they were well preserved, and the internal elastic Iamina was severely fragmented. In the arterial intima, in which the organization of fihri~~oid substance had been completed, celIulo~brous thickening was seen, and elastic and collagen fibers were formed.
In the early stage of the plasmatic arterionec~osislike lesions of the hypertensive rat mesenteric arteries, endothelial cells were rich in organelles, especially rough-surfaced endoplasmic reticulum, mitochondria, and ribosomes. Filaments, dense bodies, and dense attachments were observed in the basal part of the endothelial cytoplasm. Fibrinoid substance beneath endothelial cells showed decreased electron density in the preceding stage of its organization when intimal cells had not yet appeared in the subendothelium. Here were formed basement membranelike substance and, rarely, elastin. of the organization Intimal cells that appeared in the intima at the beginning of fibrinoid substance exhibited the appearance resembling the endothelial cells. They were modified smooth muscle cells with many cell organelles and a small
DERIVATION
OF
INTIMAL
CELLS
407
FIG. 4. Dissolution and disappearance of the intimal fibrinoid substance. Intimal smooth muscle ceMs ( SM ) are increased beneath the endothelial cells (E ), and both cell types are morphologically similar, containing filaments in their ectopIasm. Fibrinoid substance consisting mainly of fibrin (F) has mostly been dissolved and disappeared. Mesenteric artery of a hypertensive rat 10 weeks after bilateral renal artery constriction. (X5320.)
amount of myofilaments in their ectoplasm. Fibrinoid substance around the intimal cells was lowered in electron density and turned into amorphous substance of medium electron density, indicating the tendency of disappearance (Fig. 4). In the organization of fibrinoid substance, smooth muscle cell-like endothelial cells having myofilaments in their ectoplasm fell into the intima in their large part (Fig. 5) or were covered by adjacent endothelial cells presenting a picture of incorporation into the intima (Fig. 6). In the upper layer of the intima covered with the smooth muscle cell-like endothelial cells, the modified smooth muscle cells showing the appearance closely similar to the endothelial cells were found numerously. When fibrinoid substance had been completely organized, the endothelial cell junctions became closed, and desmosomes and interdigitaiton were seen there, Just beneath the endothelial cells and around the intimal smooth muscle cells were produced basement membrane and elastic and collagen fibers, and intimal smooth muscle cells turned into typical smooth muscle cells.
40s
SUZUKI,
HORI
AND OONEDA
FIG. 5. Falling of endothelial cells into the intima. Large part of an endothelial cell (E) seen in the center is buried in the intima (IN ). Filaments are increased in ectoplasm of endothelial cells (E ), which have thus turned into smooth muscle cell-like cells. Mesenteric artery of a hypertensive rat 8 weeks after bilateral renal artery constriction. ( x6250.)
In the arteries with fibrinoid degeneration, in which intimal cells were either absent or found in one or two layers beneath the eI~dothelium, endothelial cells, intimal cells, medial muscle cells, and adventitial cells were labeled at 1530 min after the administration of 3H-proline (Fig. 7). At l-2 hr, silver grains were found not only over endothelial, intimal, medial and adventitial cells but also over extracellular connective tissue. At 24 hr, the silver grains were seen more numerously over extracellular space than over the cells (Fig. 8). There was no difference in the radioactivity of the endothelial, intimal, medial, and adventitial cells.
At 30 min after 3H-proline injection, silver grains were observed over cytoplasm, especially over rough-surfaced endoplasmic reticulum and Golgi apparatus of endothelial cells and intimal smooth muscle cells, but in extracellular space they were small in amounts (Figs. 9-11). At 2 hr, the silver grains were found
DERIVATION
FIG. 6. Falling of endothelial exposes a part of its cytoplasm in the intima. In the cytoplasm is seen around the cell. Smooth a hypertensive rat 8 weeks after
OF
INTIMAL
CELLS
cells into the intima. One of several endothelial (arrow) into the lumen ( L), while the remaining of this cell, myofilaments increase and basement muscle cells are present in intima (IN). Coronary bilateral renal artery constriction. (X5540.)
409
cells (E ) is buried membrane artery of
not only over the cytoplasm of endothelial cells and intimal smooth muscle cells hut also over extracellular microfibrils, collagen fibrils, and other electron dense substance ( Fig. 12) DISCUSSION Various experiments have been performed to elucidate the derivation of intimal cells that increase in arteriosclerosis. The following cells have been considered as the origin of intimal cells: endothelial cells (Mehrotra, 1953; Haust et al., 1960; Takeda, 1961; Ooyama, 1962; Suto, 1964; Sekiguchi, 1964; Kojimahara et al., 197fa,b), medial smooth muscle cells (Filshie and Scott, 1958; Constantinides et al., 1958; Buck, 1961; Luginbiihl and Johes, 1965; Murray et al., 1966; Parker and Odland, 1966; Wissler, 1967; Jurukova and Rohr, 1968; Aikawa and Koletsky, 1970), and circulating blood cells (O’Neal et al., 1964; Halpert et al., 1966; Still et al., 1967). There is also a view that the prohferation of smooth muscle cells, inherent to the intima, may result in the intimal thicken-
410
SUZUKI,
HORI
AND
OONEDA
Fm. 7. Light microscopic autoradiograph 30 min after intravenous administration of 3Hproline. Silver grains are numerously found over endothelial cells, intimal cells lying just under the endothelium, and medial smooth muscle cells, but few are found in the intercellular space. Mesenteric artery of a hypertensive rat 9 weeks after bilateral renal artery constriction. F, fibrinoid substance in the intima; M, media.
ing ( Meessen, 1964). Some authors ( Sawatari, 1966; Suzuki, 1967; Borgers et al., 1973; Ooneda et al., 1973a) assumed that not any one special kind of cells exclusively but medial smooth muscle cells, endothelial cells, circulating blood cells, and proper intimal cells might inclusively participate in the development of the intimal thickening.
FIG. 8. Light microscopic autoradiograph 24 hr after intravenous administration of SHproline. Silver grains are more numerous over the intercellular space than over endothelial cells, intimal cells, and medial muscle cells. Mesenteric artery of a hypertensive rat 16 weeks after bilateral renal artery constriction.
DERIVATION
OF
INTIMAL
CELLS
FIG. 9. Electron microscopic autoradiograph 30 min after intravenous injection proline. Silver grains are localized over the ribosomes of an endothelial cell. Mesenteric of a hypertensive rat 15 weeks after bilateral renal artery constriction. ( X29,600.)
411
of ‘Hartery
Since intimal cells are not noticed in the rat mesenteric arteries except in their bifurcation areas, it is hardly assumable that these cells may in any other areas be involved in the organization of fibrinoid substance that deposits in the arterial intima of hypertensive rats. As to the derivation of intimal cells from circulating blood cells, the present experiments gave no conclusive evidences. It is, however, impossible to deny their penetration into the intima since the presence of endothelial cells has been confirmed electron microscopically in circulating blood, though in small number (Gaynor et al., 1970). The organization of fibrinoid substance in arterial intima began just beneath the endothelial cells (Figs. 1 and 3)) and little evidence suggested that medial muscle cells might have contributed to the organization of intimal fibrinoid substance. Occasionally, however, there was a histological finding that is indicative of this possibility. In such a case, medial muscle cells were usually well preserved, and moreover, the internal elastic lamina was fragmented on many occasions. When the medial cells were damaged so severely that the cells became invisible, it was rare that fibrinoid substance in the intima was organized from the medial side. From these facts it cannot be considered that medial smooth muscle cells play a significant role in the organization of intimal fibrinoid substance. Sekiguchi ( 1964)) who administered 3H-thymidine to rats during the healing process of fibrinoid degeneration after removing the constricting clamps from the bilateral renal arteries, found that endothelial cells were labeled at a markedly high rate, and furthermore he confirmed the transition of these labeled
412
SUZUKI,
NOR1
AND
OONEDA
FIG. 10. Electron microscopic autoradiograph 30 min after intravenous injection of “Hproline. Silver grains are seen over the Colgi complex of an intimal smooth muscle cell. hksenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. (x22,100.)
endothelial cells to intimal cells. Ooyama (1962) observed electron microscopically the organizing process of fibrinoid substance in the healing experiment of Sekiguchi and found the increase of myofilamentlike structures in the ectoplasm of endothelial cells and the presence, in the subendothelium, of the endothelial cell-like intimal cells, which he considered to have been derived from the endothelial cells. Similar findings were aIso observed by Kojimahara et al. (1971a,b) after giving antihypertel~sive drugs to hypertensive rats. The present experiments disclosed the increase of myo~lament-like structures, dense attachments, and dense bodies in the periphery of cytoplasm of endothelial cells during the organization of fibrinoid substance (Figs. 4-6); and there was a picture suggesting the falling of these smooth muscle cell-like endothelial cells into the intima (Figs. 5 and 6) or their incorporation into the intima by the covering of adjacent endothelial cells; moreover, intimal cells that mo~hologically resembled the above-mentioned smooth muscle cell-like endothelial cells were increased in the intima (Fig. 4). It was considered from these findings that the cells that increased in the subendothelium of the intima showing deposition of fibrinoid substance might have been derived from endothelial cells. It seemed that during the organization of fibrinoid substance, endothelial cells might have become smooth muscle cell-like at their own sites and turned into intimal cells when they fell into the intima.
DERIVATION
OF INTIMAL
CELLS
FIG. 11. Electron microscopic autoradiograph 30 min after intravenous injection of “H-pro&xxx Silver grains are noticed over the rough-su~aced endopl~mic re~clll~ of an intimal smooth muscle cell. Mesenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. ( X 14,770. )
In the arteries that showed deposition of fibrinoid substance, but no neu~ophil infiltration in the intima, media, and adventitia, and exhibited fibrosis of the adventitial granulation tissue, ground substance and elastic fibers were seen in the subendothelium even though intimal cells had not yet appeared there (Fig. 2). Electron microscopy revealed the presence of basement membrane, elastin, and microfibrils directly beneath endothelial cells. At this stage, the intima contained fibrinoid substance but not any intimal cells. These facts suggest that basement membrane, elastin, and microfibrils may have been produced by endo~elial cells. It is said that intimal and medial smooth muscle cells produce connective tissue protein. Karrer (1961) reported in an electron microscopic study on the rat aorta that medial mu&e cells produced collagen and elastic fibers. Ross (1971) and Fisher-Dzoga et al. (1973) suggested, by tissue culture of aortic tissue, that medial smooth muscle cells elaborated elastic fibers. Ross and Klebanoff ( 1971) , who gave prepubertal rats estradiol and then intravenous injection of 3H-proline and who investigated the aortas light and electron microscopically and autoradiographically, suggested the production of collagen and elastin by medial smooth muscle cells. Kunz et al. (1967) injected 3Hproline and 35S-sulfate to rats after constricting the aorta at two sites and observed that colIagen and mucopolysaccharides were produced by intimal cells between the ligated segment. In the present experiments, the injected 3H-proline was seen to be localized in endothelial, intimal, medial, and adventitial cells (Fig. 7). Early after 3H-
414
SUZUKI,
HORI
AND
OONEDA
FIG. 12. Electron microscopic a~ltoradi~~aph 2 hr after intravenous injection of “H-proline. Silver grains are seen over the collagen fibrils (A) and extracellular electron dense s&stance (B) in the intima. Mesenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. (A and B: X48,000. ) proline illjectiorl (at 15-30 mm), it was found abundantly within the cell (Fig. 7), but thereafter it was abundant in estracellular space (Fig. 8). Electron microscopic autoradiography demonstrated that the silver grains were seen over the cell organelles or cystoplasm of endothelial cells and intimal and medial smooth muscle cells (Figs. 9-11). It can therefore be assumed that endothelial cells and intimal and medial smooth muscle cells may produce elastin and collagen. The intimal cells resembled the endothelial cells both nlorpholo~i~ally and functionally. Moreover, there was a picture indicating that the latter might have been incorporated or fallen into the intima (Figs. 5 and 6). From these facts it is considered that the cells involved in the organization of fibrinoid substance are mostly endothelial cells and partly medial smooth muscle cells.
ACKNOWLEDGMENTS The authors are technical assistance.
grateful
to Mr.
T.
Fukushima,
Miss
E. Yoshida,
and
Miss
M.
Saito
for
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DERIVATION
OF
INTIMAL
CELLS
415
BORGERS, M., SCHAPER, J., and SCHAPEH, W, (1973). Th e origin of subendothelial cells in developing coronary collaterals. A cytochemical approach. Virchows Arch. A 358, 281-294. BUCK, R. C. ( 1961). Intimal thickening after ligature of atteries. Circ. Res. 9, 418426. CONSTANTINIDES, P., GUTMANN-AUERSPERG, N., and HOPES, D. ( 1958). Acceleration of intimal atherosclerosis through prior medial injury. Arch. P~hol, 66, 247-254. FILSHXE, I., and SCOTT, G. B. D. ( 1958). The organization of experimental venous thrombi. J. Pathol. Bacterial. 76, 71-76. FISHER-DZOGA, K., JONES, R. M., VESSELXNOVITCH, D., and WISSLER, R. W. (1973). Ultrastructural and ~muno~stochemic~ studies of primary culture of aortic medial cells. Exp. &foE. PathoE. 18, 162-176. GAYNOR, E., BOUVIER, C. A., and CINTRON, J. R. (1970). A technique for locating rare cells in the circulation for ultramicroscopy. PTOC. Sot. Exp. Biol. Med. 133, 520-523. HALPERT, B., O’NEAL, R. M., JORDAN, G. L., JR., and DEBAKEY, M. E. ( 1966). “Vasa vasornm” of dacron prosthesis in canine aorta. Arch. P~ho~. 81, 412-417. HAUNT, M. D., MORE, R. H., and MOVAT, H. Z. (1960). The role of smooth muscle cells in the fibrogenesis of arteriosclerosis, Amer. J. Pathol. 37, 377-390. JURUXOVA, Z., and ROHR, H. P. ( 1968). Beitrag zur Bildung bindegewebiger Matrix in glatten Muskelzellen. El~ktronenmikroskopisch-autoradiographische Untersuchungen mit “S-S&at an glatten Muskelzellen nach Doppelligatur der Arteria carotis. PathoE. Eur. 3, 551570. KARRER, H. E. (1961). An electron microscope study of the aorta in young and in aging mice. J. Vltrastruct. Res. 5, 1-27. KOJIMAHARA, M. (1967). Healing and exacerbation of arterial lesions in rats with experimental hypertension, with special reference to effects of antih~e~ensive drugs on arterial lesions. Gunma J. Med. Sci. 16, l-42. KOJIMAHARA, M., SEKIYA, K., and OONEDA, G. (1971a). Studies on the healing of arterial lesions in experimental hypertension. An electron microscopy study of the healing process of intimal fibrinoid degeneration in hypertensive rats. V~rchows Arch. A 354, 150-160. KOJXMAHARA, M., SEKIYA, K., and OONEDA, G. (1971b). Studies on the healing of arterial lesions in experimental hypertension. An electron microscopy study on the origin of intimal smooth muscle cells in the intima with marked dysoria. Virchows Arch. A 354, 161-168. KUNZ, J., KRANZ, D., and KEIM, 0. ( 1967). Autoradio~raphische Untersuchungen zur Synthese von DNS, Kollagen und ~~u~polysaccha~den bei der experimentellen Proliferation der Aortenintima. Virchows Arch. Pathol. Anat. 342, 345-352. LUGINBH~~L, H., and JOHES, J. E. T. ( 1965). The morphology and morphogenesis of atherosclerosis in aged swine. Ann. N.Y. Acad. Sci. 127, 763-779. MEESSEPU’, H. (1944). Uber den pl~~lichen Herztod bei Friihsklerose und Fr~hthrombose der Komnalarterien bei Mtinern unter 45 Jahren. Z. Kreislautforsch. 36, 185-201. MEHROTRA, R. M. L. ( 1953). An experimental study of the changes which occur in ligated arteries and veins. J. Pathol. Bacterial. 65, 307313. MURRAY, M., SCHROIX, G. R., and BERG, H. F. (1966). Role of smooth muscle cells in healing of injured arteries. Arch. Pathol. 82, 138-146. O’NEAL, R. M., JORDAN, G. L., JR., RABIN, E. R., DEBAKEY, M. E., and HALPERT, B. (1964). Cells grown on isolated intravascuhr dacron hub; an electron microscopic study. Exp. Mol. Pathol. 3, 403-412. OONEDA, G., YOSHI~A, Y., and SUZUKI, K. (1973a). Morphogenesis of arteriosclerosis: Proliferation and insudation. Jap. J. Atheroscler. 1, 3-13 (in Japanese). OONEDA, G., YOSHIDA, Y., SUZUKI, K., and SEKIGUCHI, T. (1973b). Morphogenesis of plasmatic arterionecrosis as the cause of hypertensive intracerebral hemorrhage. Virchows Arch. A 361, 3138. OOYAMA, Y. (1962). Electron microscopy studies of arterial lesions, with special reference to the morphogenesis of arteriosclerosis and fibrinoid degeneration. Trans. Sot. Pathol. Jap. 51, 146-177 (in Japanese). PAR~P,, F., and ODLAND, G. F. ( 1966}. A correlative histochemical, biochemical and electron microscopic study of experimental atherosclerosis in the rabbit aorta with special reference to the myointimal cell. Amer. J. Pathol. 48, 197-239. Ross, R., and KLEBANOFF, S. ( 1971). The smooth muscle cell. I. In vivo synthesis of connective tissue proteins. J. Cell Biol. 50, 159-171.
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Ross, R. (1971). The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell Biol. 50, 172-186. SAWATARI, M. (1966). Electron microscopic studies on arteriosclerosis and arterial fibrinoid degeneration. Gunma J. Med. Sci. 15, 229-303. SEKIGIJCHI, M. (1964). The development and healing of arterial lesions in hypertensive rats with bilaterally constricted renal arteries. Kitakanto Med. Sci. 14, l-35 (in Japanese). STILL, W. J. S., GHANI, A. R., and DENNISON, S. M. ( 1967). The organization of isolated mural thrombi in aortic grafts. Amer. J. Pathol. 51, 1013-1029. SUTO, H. (1964). Experimental studies on arterial lesions. 111. Autoradiographic studies on the morphogenesis of arteriosclerosis using thymidine-“H. Kitakanto Med. Sci. 14, 141-185 ( in Japanese ) . Suzum, K. ( 1967). Experimental studies on morphogenesis of arteriosclerosis, with special reference to relation between hemodynamic change and developments of cellulofibrous intimal thickening and atherosclerosis. Gunma J. Med. Sci. 16, 185-243. SUZUKI, K., OONEDA, G., and OOKAWARA, S. (1972). On the intimal cells derived from endothelial cells in the spontaneous healing of hypertensive arterial lesions. J. Jap. CoZZ. Angiol. 12, 93-97 (in Japanese). TAKATAMA, M. ( 1960). Morphogenesis of arterial lesions, especially fibrinoid degeneration, in hypertensive rats with surgicaRy constricted renal arteries. Trans. Sot. Pathol. Jap. 49, 494-526 (in Japanese). TAKEDA, F. ( 1961). Morphogenesis of arteriosclerosis. Pathological studies of the acrylic resin-invested common carotid artery of the rabbit. Trans. Sot. Pathol. Jup. 50, 367393 ( in Japanese ) . smooth muscle or multifunctional WISSLER, R. W. ( 1967). The arterial media cell, mesenchyme? Circulation 36, 14.
Ah‘D
Derivation
MOLECULAR
23, 402-416
( 1975)
of Intimal Cells Appearing in Spontaneous Process of Hypertensive Arterial Lesions KEIJI
Second
PATHOLOGY
SUZUKI,
Department
Received
SADAO
Honr,
AND
GENJU
of Pathology, School of Medicine, Maebashi, Japan March
20> 197.5,
and in revised
form
Healing
OONE~A Gunma
May
Univekty,
20, 1975
Light and electron microscopic and autoradiographic studies were made on the process of spontaneous organization of fibrinoid substance deposited in the intima, that is, spontaneous healing of intimal fibrinoid degeneration of the hypertensive rat mesenteric arteries at 8-24 weeks after the constriction of the bilateral renal arteries. The organization of the intimal fibrinoid substance by intimal cells was performed mainly from the luminal side and infrequently from the medial side. Indigenous intimal cells and circulating blood cells were nat positively involved in the organization. During the organization of fibrinoid substance, the endothelial cetls turned into smooth muscle cell-like cells in situ, and there was a picture s~l~~esting the migration of these smooth muscle cell-like endothehal rells into the intima or their incorporation into the intima by the covering of adjacent endothelial cells. Experiments with intravenous administration of W-proline revealed that endothelial cells and intimal and medial smooth muscle cells resembled each other functionally. It can therefore be said that the majority of the cells that participate in the organization of intimal fibrinoid substance are intimal smooth muscle cells derived from endothelial cells, and a part of the cells is derived from medial smooth muscle cells. The cellulofibrous tissue of the intima after the complete organization of intimal fibrinoid substance resulted from the proliferation of the en(~~tIlelial cellderived intimal smooth muscle cells and from the fiber and ground substance formation by these.
INTRODUCTION There are many theories concerning the derivation of smooth muscle cells constituting arterial intimal thickening, and no consensus has yet been attained, Among them are found the following: ( 1) medial smooth muscle cell-derivation theory (Filshie and Scott, f958; Consta~tinides et al, 1958; Buck, 1961; Luginbiihl and Johes, 1965; Murray et al., 1966; Parker and Odland, 1966; Wissler, 1967; Jurukova and Rohr, 1968; Aikawa and Koletsky, 1970); (2) endothelial cellderivation theory (Mehrotra, 1953; Haust et al., 1960; Takeda, 1961; Ooyama, 1962; Suto, 1964; Sekiguchi, 1964; Kojimahara et al., 197&b); (3) circulating blood cell-derivation theory ( O’Neal et al., 1964; Halpert et al., 1966; Still et al., 1967); (4) in t imal cell-derivation theory (Meessen, 1964); and (5) plural cell derivation theory, which asserts that not any single kind but plural kinds of cells, such as medial smooth muscle cells, endothelial cells, proper intimal 402 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any farm reserved.
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smooth muscle cells, and circulating blood cells, can be the origin of the increased intimal cells, contributing to intimal thickening (Sawatari, 1966; Suzuki, 1967; Borgers et al., 1973; Ooneda et al., 1973a). Fibrinoid substance, deposited in the arterial intima of experimental hypertensive rats, was organized to form cellulofibrous intimal thickening spontaneously (Takatama, 1960; Suzuki et al., 1972) or after removing the constricting clamps from the renal arteries (Se~~chi, 1964) or after giving antihypertensive drugs (Kojimahara, 1967; Kojimahara et al., 1971a,b). The lesions represented a kind of arteriosclerosis. In the present study, spontaneous organizing process of intima1 fibrinoid substance, i.e., spontaneous healing of intimal fibrinoid degeneration, in the mesenteric and coronary arteries of hypertensive rats was investigated at 8-24 weeks after bilateral renal artery constriction by light and electron microscopy and autoradiography, and the derivation of intimal cells, which increased in the intima, was studied. MATERIAL
AND METHODS
Hypertension was experimentally induced in 61 male Wistar rats, weighing 60-80 gm, by constricting the bilateral renaI arteries with silver clamps 0.23 mm in internal diameter. The animals were sacrificed at 842 postoperative weeks, when their systolic blood pressure was above 160 mm Hg and nodular lesions were found in the mesenteric arteries. Light Microscopic Observation Thirty rats were laparotomized under ether anesthesia, and a lower part of the abdominal aorta was cut to kill them by bleeding. After the sacrifice, the mesenteric arteries showing nodular lesions were taken out and fixed with 1070 buffered neutra1 formalin. After the tissues were dehydrated with graded ethanol and embedded in paraffin, thin sections were prepared, which were stained with HE, Mallory, Weigert’s elastic fiber, and Alcian blue stains. Electron Microscopic Observation Twenty rats were laparotomized under ether anesthesia, the mesenteric arteries and abdominal aorta were exposed, and the latter was clamped above and below the arising sites of the former to stop blood flow. Then a 170 osmic acid was injected into this aortic segment to perfuse the mesenteric arteries with it, Thereafter, the mesenteric arteries with nodular lesions were removed and refixed with a 1% osmic acid for 2 hr. After dehydration and embedding with Epon 812, ultrathin sections were prepared and double stained with uranyl acetate and lead hydroxide to be observed electron microscopically. The coronary arteries treated similarly were also examined by electron microscopy in five animals. Light Microscopic Autoradiographic
Observation
Seven hypertensive rats with well-developed mesenteric nodular lesions were injected with 3H-prohne ( L-xH-proline, specific activity: 3.02 Ci/mmole, New England Nuclear Corporation, Boston, Mass. ) at a dose of 5 #&‘gm body
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weight through the femoral vein. At 5, 15, 30, 60, and 120 min thereafter, a proximal and a distal arterial part of the noduIar lesions were ligated, and then the intervening arterial segments were removed from four animals. Moreover, at 4, 6, and 24 hr after the intravenous injection of 3H-proline, a nodular arterial segment was taken off from each of three animals. After formalin fixation, paraffin sections were prepared and deparaffinized and then coated with Sakura autoradiograph emulsion NR-M2 (Konishiroku Photo Ind. Co., Ltd., Tokyo) to be exposed to irradiation in a shaded box for 2-3 weeks. After development with Konidol X (KoI~ish~oku Photo Ind. CO., Ltd., Tokyo) and fixation with Konifix (Konishiroku Photo Ind. CO., Ltd., Tokyo), the sections were subjected to nuclear staining with hematoxylin and observed light microscopically. Electron Microscopic Autodiographic
Observation
Four animals were injected with “H-proline (10 ~Ci/gm) mto the femoral vein and, at 30 and 120 min thereafter, the nodular arterial lesions were removed. They were fixed with a 1% osmic acid for 2 hr and then dehydrated and embedded with Epon 812. Ultrathin sections from these tissues were coated with Sakura autoradiographic emulsion NR-H2 (Konishiroku Photo Ind. Co., Ltd., Tokyo), exposed to irradiation for l-2 months, developed with Konidol X and fixed with Konifix, and double stained with uranyl acetate and lead hydroxide to be examined electron microscopically.
The nodular arterial lesions under the light microscope proved to be very similar to the plasmatic arterionecrosis ( Ooneda et al., 197310). Early and late lesions were seen in the rat mesenteric arteries at S-16 postoperative weeks. After 16 weeks, most of them were organized. Eady lesions. Numerous neutrophils were attached to the endotheliai surface. The intima was infiltrated with neutrophils, and plasma ~onlponents were accumulated in it. Medial smooth muscle cells became necrotic, and numerous neutrophils and nuclear fragments were seen in the media. In the adventitia there were marked proliferation of fibroblasts and conspicuous neutrophil infiltration. In the arteries with abundant accuInulation of fibrinoid substance in the intima, the attachment of neutrophils to the endothelial surface was not seen, and endothelial cells turned rather flat with basophilic cytoplasm, nuclear chromatin being increased. The fibrinoid substance in the intima was stained red with HE stain and seen as the accumulation of trabecular or rod-shaped substances. Neither the intima nor the media showed marked neutrophil in~ltratio~l. Generally in the arteries showing fibrinoid necrosis the media became atrophic and the medial cells were reduced or even absent, The nuclei of remaining medial cells were enlarged and their cytoplasm became basophilic, but the migration of the medial smooth muscle cells into the intima was not noticed. Organized Lesions.Endothelial cells were enlarged and proliferated, showing rather basophilic cytoplasm and increased nuclear chromatin. In the early stage of the orgal~ization, the staining property of the subendothelial fibrinoid sub-
DERIVATION
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FIG. 1. Early stage of organization of intimal fibrinoid substance in the mesenteric artery. Intimal cells are seen just beneath the endotheliar cells, which cover the fibrinoid substance (F) deposited in the intima. Migration of the medial cells into the intima is not seen. Necrotic change of the medial smooth muscle cells is conspicuous. Hypertensive rat 12 weeks after bilateral renal artery constriction. M: media. HE stain.
stance was decreased, Intimal cells appeared beneath the endothelium, and the fibrinoid substance around the intimal cells disappeared (Fig. 1). Weigert’s elastic stain revealed newly formed granular and fibrillar elastic fibers just beneath the endothelial cells and around the intimal cells (Fig. 2). A large amount of acid mucopolysaccharides stained with Alcian blue was noticed around the intimaI cells and beneath the endothelial cells.
FIG. 2. In the intima with fibrinoid substance (F), newly formed granular elastin (arrow) is seen directly beneath the endothelial cells. Mesenteric artery of a hypertensive rat 12 weeks after bilateral renal artery constriction. Weigert’s elastic fiber stain.
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FIG. 3. Organization of intimal fibrinoid .&stance. Intimal cells are increased in the inner intima, while fibrinoid substance (F) remains in the deep intima. The organization from the media (M) is not marked. hlesenteric artery of a hypertensive rat 12 weeks after hilateral renal artery constriction. HE stain. The intimal fibrinoid substance was organized from the luminal side, and intimal cells that proliferated in the subendothelium entered into the fibrinoid substance, thus decreasing its staining property around them. When the organization advanced, intimal cells were markedly proliferated, often presenting mitotic figures, and elastin and ground substance were formed between the cells, fibrinoid substance remaining only in the deep intima (Fig. 3). It was infrequent that the organization of intima1 fibrinoid substance was performed from the medial side. Thus, on rare occasions, intimal fibrinoid substance was penetrated by medial smooth muscle cells, when they were well preserved, and the internal elastic Iamina was severely fragmented. In the arterial intima, in which the organization of fihri~~oid substance had been completed, celIulo~brous thickening was seen, and elastic and collagen fibers were formed.
In the early stage of the plasmatic arterionec~osislike lesions of the hypertensive rat mesenteric arteries, endothelial cells were rich in organelles, especially rough-surfaced endoplasmic reticulum, mitochondria, and ribosomes. Filaments, dense bodies, and dense attachments were observed in the basal part of the endothelial cytoplasm. Fibrinoid substance beneath endothelial cells showed decreased electron density in the preceding stage of its organization when intimal cells had not yet appeared in the subendothelium. Here were formed basement membranelike substance and, rarely, elastin. of the organization Intimal cells that appeared in the intima at the beginning of fibrinoid substance exhibited the appearance resembling the endothelial cells. They were modified smooth muscle cells with many cell organelles and a small
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FIG. 4. Dissolution and disappearance of the intimal fibrinoid substance. Intimal smooth muscle ceMs ( SM ) are increased beneath the endothelial cells (E ), and both cell types are morphologically similar, containing filaments in their ectopIasm. Fibrinoid substance consisting mainly of fibrin (F) has mostly been dissolved and disappeared. Mesenteric artery of a hypertensive rat 10 weeks after bilateral renal artery constriction. (X5320.)
amount of myofilaments in their ectoplasm. Fibrinoid substance around the intimal cells was lowered in electron density and turned into amorphous substance of medium electron density, indicating the tendency of disappearance (Fig. 4). In the organization of fibrinoid substance, smooth muscle cell-like endothelial cells having myofilaments in their ectoplasm fell into the intima in their large part (Fig. 5) or were covered by adjacent endothelial cells presenting a picture of incorporation into the intima (Fig. 6). In the upper layer of the intima covered with the smooth muscle cell-like endothelial cells, the modified smooth muscle cells showing the appearance closely similar to the endothelial cells were found numerously. When fibrinoid substance had been completely organized, the endothelial cell junctions became closed, and desmosomes and interdigitaiton were seen there, Just beneath the endothelial cells and around the intimal smooth muscle cells were produced basement membrane and elastic and collagen fibers, and intimal smooth muscle cells turned into typical smooth muscle cells.
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FIG. 5. Falling of endothelial cells into the intima. Large part of an endothelial cell (E) seen in the center is buried in the intima (IN ). Filaments are increased in ectoplasm of endothelial cells (E ), which have thus turned into smooth muscle cell-like cells. Mesenteric artery of a hypertensive rat 8 weeks after bilateral renal artery constriction. ( x6250.)
In the arteries with fibrinoid degeneration, in which intimal cells were either absent or found in one or two layers beneath the eI~dothelium, endothelial cells, intimal cells, medial muscle cells, and adventitial cells were labeled at 1530 min after the administration of 3H-proline (Fig. 7). At l-2 hr, silver grains were found not only over endothelial, intimal, medial and adventitial cells but also over extracellular connective tissue. At 24 hr, the silver grains were seen more numerously over extracellular space than over the cells (Fig. 8). There was no difference in the radioactivity of the endothelial, intimal, medial, and adventitial cells.
At 30 min after 3H-proline injection, silver grains were observed over cytoplasm, especially over rough-surfaced endoplasmic reticulum and Golgi apparatus of endothelial cells and intimal smooth muscle cells, but in extracellular space they were small in amounts (Figs. 9-11). At 2 hr, the silver grains were found
DERIVATION
FIG. 6. Falling of endothelial exposes a part of its cytoplasm in the intima. In the cytoplasm is seen around the cell. Smooth a hypertensive rat 8 weeks after
OF
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cells into the intima. One of several endothelial (arrow) into the lumen ( L), while the remaining of this cell, myofilaments increase and basement muscle cells are present in intima (IN). Coronary bilateral renal artery constriction. (X5540.)
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cells (E ) is buried membrane artery of
not only over the cytoplasm of endothelial cells and intimal smooth muscle cells hut also over extracellular microfibrils, collagen fibrils, and other electron dense substance ( Fig. 12) DISCUSSION Various experiments have been performed to elucidate the derivation of intimal cells that increase in arteriosclerosis. The following cells have been considered as the origin of intimal cells: endothelial cells (Mehrotra, 1953; Haust et al., 1960; Takeda, 1961; Ooyama, 1962; Suto, 1964; Sekiguchi, 1964; Kojimahara et al., 197fa,b), medial smooth muscle cells (Filshie and Scott, 1958; Constantinides et al., 1958; Buck, 1961; Luginbiihl and Johes, 1965; Murray et al., 1966; Parker and Odland, 1966; Wissler, 1967; Jurukova and Rohr, 1968; Aikawa and Koletsky, 1970), and circulating blood cells (O’Neal et al., 1964; Halpert et al., 1966; Still et al., 1967). There is also a view that the prohferation of smooth muscle cells, inherent to the intima, may result in the intimal thicken-
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Fm. 7. Light microscopic autoradiograph 30 min after intravenous administration of 3Hproline. Silver grains are numerously found over endothelial cells, intimal cells lying just under the endothelium, and medial smooth muscle cells, but few are found in the intercellular space. Mesenteric artery of a hypertensive rat 9 weeks after bilateral renal artery constriction. F, fibrinoid substance in the intima; M, media.
ing ( Meessen, 1964). Some authors ( Sawatari, 1966; Suzuki, 1967; Borgers et al., 1973; Ooneda et al., 1973a) assumed that not any one special kind of cells exclusively but medial smooth muscle cells, endothelial cells, circulating blood cells, and proper intimal cells might inclusively participate in the development of the intimal thickening.
FIG. 8. Light microscopic autoradiograph 24 hr after intravenous administration of SHproline. Silver grains are more numerous over the intercellular space than over endothelial cells, intimal cells, and medial muscle cells. Mesenteric artery of a hypertensive rat 16 weeks after bilateral renal artery constriction.
DERIVATION
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FIG. 9. Electron microscopic autoradiograph 30 min after intravenous injection proline. Silver grains are localized over the ribosomes of an endothelial cell. Mesenteric of a hypertensive rat 15 weeks after bilateral renal artery constriction. ( X29,600.)
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Since intimal cells are not noticed in the rat mesenteric arteries except in their bifurcation areas, it is hardly assumable that these cells may in any other areas be involved in the organization of fibrinoid substance that deposits in the arterial intima of hypertensive rats. As to the derivation of intimal cells from circulating blood cells, the present experiments gave no conclusive evidences. It is, however, impossible to deny their penetration into the intima since the presence of endothelial cells has been confirmed electron microscopically in circulating blood, though in small number (Gaynor et al., 1970). The organization of fibrinoid substance in arterial intima began just beneath the endothelial cells (Figs. 1 and 3)) and little evidence suggested that medial muscle cells might have contributed to the organization of intimal fibrinoid substance. Occasionally, however, there was a histological finding that is indicative of this possibility. In such a case, medial muscle cells were usually well preserved, and moreover, the internal elastic lamina was fragmented on many occasions. When the medial cells were damaged so severely that the cells became invisible, it was rare that fibrinoid substance in the intima was organized from the medial side. From these facts it cannot be considered that medial smooth muscle cells play a significant role in the organization of intimal fibrinoid substance. Sekiguchi ( 1964)) who administered 3H-thymidine to rats during the healing process of fibrinoid degeneration after removing the constricting clamps from the bilateral renal arteries, found that endothelial cells were labeled at a markedly high rate, and furthermore he confirmed the transition of these labeled
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FIG. 10. Electron microscopic autoradiograph 30 min after intravenous injection of “Hproline. Silver grains are seen over the Colgi complex of an intimal smooth muscle cell. hksenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. (x22,100.)
endothelial cells to intimal cells. Ooyama (1962) observed electron microscopically the organizing process of fibrinoid substance in the healing experiment of Sekiguchi and found the increase of myofilamentlike structures in the ectoplasm of endothelial cells and the presence, in the subendothelium, of the endothelial cell-like intimal cells, which he considered to have been derived from the endothelial cells. Similar findings were aIso observed by Kojimahara et al. (1971a,b) after giving antihypertel~sive drugs to hypertensive rats. The present experiments disclosed the increase of myo~lament-like structures, dense attachments, and dense bodies in the periphery of cytoplasm of endothelial cells during the organization of fibrinoid substance (Figs. 4-6); and there was a picture suggesting the falling of these smooth muscle cell-like endothelial cells into the intima (Figs. 5 and 6) or their incorporation into the intima by the covering of adjacent endothelial cells; moreover, intimal cells that mo~hologically resembled the above-mentioned smooth muscle cell-like endothelial cells were increased in the intima (Fig. 4). It was considered from these findings that the cells that increased in the subendothelium of the intima showing deposition of fibrinoid substance might have been derived from endothelial cells. It seemed that during the organization of fibrinoid substance, endothelial cells might have become smooth muscle cell-like at their own sites and turned into intimal cells when they fell into the intima.
DERIVATION
OF INTIMAL
CELLS
FIG. 11. Electron microscopic autoradiograph 30 min after intravenous injection of “H-pro&xxx Silver grains are noticed over the rough-su~aced endopl~mic re~clll~ of an intimal smooth muscle cell. Mesenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. ( X 14,770. )
In the arteries that showed deposition of fibrinoid substance, but no neu~ophil infiltration in the intima, media, and adventitia, and exhibited fibrosis of the adventitial granulation tissue, ground substance and elastic fibers were seen in the subendothelium even though intimal cells had not yet appeared there (Fig. 2). Electron microscopy revealed the presence of basement membrane, elastin, and microfibrils directly beneath endothelial cells. At this stage, the intima contained fibrinoid substance but not any intimal cells. These facts suggest that basement membrane, elastin, and microfibrils may have been produced by endo~elial cells. It is said that intimal and medial smooth muscle cells produce connective tissue protein. Karrer (1961) reported in an electron microscopic study on the rat aorta that medial mu&e cells produced collagen and elastic fibers. Ross (1971) and Fisher-Dzoga et al. (1973) suggested, by tissue culture of aortic tissue, that medial smooth muscle cells elaborated elastic fibers. Ross and Klebanoff ( 1971) , who gave prepubertal rats estradiol and then intravenous injection of 3H-proline and who investigated the aortas light and electron microscopically and autoradiographically, suggested the production of collagen and elastin by medial smooth muscle cells. Kunz et al. (1967) injected 3Hproline and 35S-sulfate to rats after constricting the aorta at two sites and observed that colIagen and mucopolysaccharides were produced by intimal cells between the ligated segment. In the present experiments, the injected 3H-proline was seen to be localized in endothelial, intimal, medial, and adventitial cells (Fig. 7). Early after 3H-
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FIG. 12. Electron microscopic a~ltoradi~~aph 2 hr after intravenous injection of “H-proline. Silver grains are seen over the collagen fibrils (A) and extracellular electron dense s&stance (B) in the intima. Mesenteric artery of a hypertensive rat 15 weeks after bilateral renal artery constriction. (A and B: X48,000. ) proline illjectiorl (at 15-30 mm), it was found abundantly within the cell (Fig. 7), but thereafter it was abundant in estracellular space (Fig. 8). Electron microscopic autoradiography demonstrated that the silver grains were seen over the cell organelles or cystoplasm of endothelial cells and intimal and medial smooth muscle cells (Figs. 9-11). It can therefore be assumed that endothelial cells and intimal and medial smooth muscle cells may produce elastin and collagen. The intimal cells resembled the endothelial cells both nlorpholo~i~ally and functionally. Moreover, there was a picture indicating that the latter might have been incorporated or fallen into the intima (Figs. 5 and 6). From these facts it is considered that the cells involved in the organization of fibrinoid substance are mostly endothelial cells and partly medial smooth muscle cells.
ACKNOWLEDGMENTS The authors are technical assistance.
grateful
to Mr.
T.
Fukushima,
Miss
E. Yoshida,
and
Miss
M.
Saito
for
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DERIVATION
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