Morphogenesis of human aortic coarctation

EXPERIMENTaL

3ND

MOLECULAR

PdTHOLOGY

Morphogenesis

6, 25-38 (1967)

of Human

Aortic

Coarctation

JOHN U. BALIS, AK Soo CHAN, AND PATRICK Deparlment

of Pathology and and

E. CONEN

Research Institute, The Hospital fur Sick The University of Toronto, Canada’ Received

August

Children,

Toronio,

12, 1965

Since Morgagni’s description in 1660, coarctation of the aorta has been recognized for over 300 years and has been thoroughly studied as a clinicopathological entity (Edwards et al., 1948; Clagett et al., 1954; Glass et,al., 1960). In contrast, there are few papers (Edwards et al., 194s; Clagett et al., 1954) which consider structural alterations of the aortic coarctation in spite of the fact that this lesion may serve as a model to study the mode of formation and progression of important arterial changes. Thus, rupture and dissecting aneurysms of the aortic wall often occur in the vicinity of the aortic coarctation (Reifenstein et al., 1947). Most important, however, this lesion and the adjacent upper and lower port’ion of the aortic wall provide an opportunity to investigate responsesof the arterial tissue to different hemodynamic factors. These factors seemto be causally related to the formation of inbimal atheromatous changes that are often observed in the vicinity of the aortic constriction (Edwards et al., 1945). The latter is due to a congenital medial lesion that is characterized by abnormal proliferation of smooth muscle cells and fibroelastic tissue. This communication describes and evaluates maturation processesthrough infancy and childhood in the congenit’al component of the aortic coarctat,ion. The inbimal lesions will be described in a separate communication. MATERIALS

AND

METHODS

Twenty-seven surgical specimenscomprising complete or incomplete segmentsof the aortic coarctation and small portions of the adjacent proximal and distal aortic wall were obtained rapidly after excision. For proper orientation, a black silk suture was placed by the surgeon in the distal portion of the specimen. The inferior wall of the aortic segment which included the ligamenturn arteriosus (or ductus arteriosus) was often left in situ. The specimen was immediately placed in 5% phosphatebuffered formalin (pH 7.4), where it was divided into t’hree pieces. The piece proximal to coarctation was labeled I; that distal to coarctation was labeled II and the coarctated area was labeled III. Rectangular blocks, 1.5 x 2 x 5 mm, were obtained from each of the three pieces and were labeled according to their location and orientation. Some of these blocks were freed of the adventitia and were subsequently postfixed for 2 hours in ice-cold Palade’s buffered osmium tetroxide solution (pH 7.4), containing 0.25 M sucrose (Caulfield, 1957). The remainder of the blocks were 1 Supported

by Canadian Medical

Research Council grant MA 1570. 25

26

J.

U.

BALIS,

A.

S. CHAN,

AND

P.

E.

CONEN

placed in Lillie’s buffered formalin (pH 7) for embedding in paraffin. The blocks fixed in osmium tetroxide were dehydrated in graded series of ethanol and embedded in Epon 812 according to the method of Luft (1961). Thin sections were cut on a Porter-Blum microtome, stained with uranyl acetate, lead acetate (Millonig, 1961), or phosphotungstic acid and examined in a Philips 200 electron microscope at original magnifications ranging from 1,000 to 20,000. In addition, 0.5-p thick sections were cut from each Epon-embedded block and stained with toluidine blue or PAS. Aortas obtained less than 2 hours after death from 18 fetuses of 6-28 weeks gestation and from 12 infants and young children were similarly processed. These aortas served as controls for the light and electron microscopic studies. The paraffin-embedded tissues were stained with hematoxylin and eosin; Verhoeff’s elastic van Gieson; periodic acid-Schiff (PAS), and Best’s carmine, with and without diastase pretreatment; alcian blue; toluidine blue; phosphotungstic acid hematoxylin; Gomori’s iron; and pentachrome I or II (Movat, 1955). Selected formalin fixed specimens were cut on a Cryostat, and 8-p thick sections were stained with oil red 0. Clinical pathological data. The clinical findings of the cases studied were usually typical of the disease and the surgical treatment consisted of resection and end-toend anastomosis. Clinical details and measurements obtained at operation are recorded in Table I. Most patients were male children. The blood pressure, systolic and diastolic, was elevated in the arms in most cases. The systolic pressure was usually recorded in the legs and was either low or unobtainable. The luminal diameter of the coarctation varied from less than 1 mm to 6 mm. The length of the coarctated portion was recorded in 12 cases and measured from 3 to 12 mm. The majority of patients showed rapid postoperative improvement and normal blood pressure readings were obtained. RESULTS Light microscopy In aortic coarctations of newborn and young infants there was no sharp demarcation between intima and media. In the area normally occupied by a continuous internal elastic lamina there were numerous elastic fibrils associated with spindleshaped cells (Fig. 1). In the midportion of the media there were few elastic fibrils and collagen but numerous spindle-shaped or stellate cells. These cells usually contained granules which gave a positive reaction with Best’s carmine and PAS stains (Fig. 2, arrows). The granules were seldom seen in intimal spindle-shaped cells. They also gradually decreased in medial cells located away from the aortic coarctation. In normal aortas of the same age group and in human fetuses older than 25 weeks gestation, PAS-positive granules were only occasionally observed, but in embryos and young fetuses they were very frequently present in medial smooth muscle cells (Chan et al., 1965). At the site of coarctation the media of the aorta appeared to develop characteristic changes with age. Thus, PAS-positive granules in smooth muscle cells gradually decreased and were observed only occasionally in aortas of children older than 5

AORTIC

TABLE CLINICAL

DATA

1* 2* 3* 4* 5* 6 7

sus;

Sex

Age

M M

6 d. 2% m. 8 m.

M

234Y.

M M

4 5 5 5 5 5 5

M

8 9 10 11*

M M M M M

12 13* 14 15 16* 17

F M M M F M

18* 19

M M

20 21* 22 23 24 25 26% 27*

M F M M

7 Y7 Y. 7 Y.

8 Y.

F

M

13 y.

Leg

85 (syst.) 200/100

6 Y. 6 Y. 6 Y. 6 Y. 6 Y.

y. y. y. y. y. y.

0 Cases with (*) have also been studied -, blood pressure not recordable.

EXAMINED

pressure

Rt. arm

Y. Y. Y, Y. Y. Y. Y.

10 11 11 12 12 12

M M

I

ON 27 CASES OF AORTIC COARCTATION LIGHT MICROSCOPY” Blood

Case No.

27

COARCTATION

170/100 170/80 133/90 150/90 145/90 120/75 150/90 160/100 x0/50 130/70 x0/90 m/95 98/80 138/98 134/m 150/90 148/78 190/130 120/80 134/90 114/70 162/110 150/60

140/90 118/60 by electron

(syst.)

Position

100 65 70 90 70 90 -

100 90 96 microscopy.

of coarct.

BY

Int. dia. of coarct (mm)

PreductalP Preductalr Postductal Juxtaductal Postductal Juxtaduct,al Postductal Postductal Postductai Postductal Postductal Postductal Postductal Postductal Postductal Postductalr Postductal Postductal Postductal Postductal Postductal Postductal Postductal PostductaJ Postductal PostductalP Postductal P, patent

0.5

1 0.5 2

1 34 3 34 4 6 3 3 4 5 4

1 2 3 2 3.5

1 6 3 0.5 3 6 ductus

arterio-

years. In addition, aggregated medial cells were increasingly associated with abundant small elastic fibers and collagen, but not with elastic lamellas (Fig. 3 and 4). The medial lesion usually involved the inner half of the media and was separated from the intima by numerous condensedelastic fibers (Fig. 3, arrow). The peripheral portions of the media often revealed normal elastic lamellas regularly interlacing with smooth muscle cells !(Fig. 3, IV). In addition, a portion of the aortic wall at the level of the coarctation was spared by the medial lesion and it was in this location that the ductus arteriosus, identified in a few instances, was found. Focal increase of “ground substance,” as judged by alcian blue, toluidine blue, and pentachrome stains, was often observed, particularly in coarctations of young children. The amount of ground substance, however, was normal or even decreased in areas of excessive collagenization, which was frequent in coarctations obtained from children older than 7 years. In this group of patients, capillaries originating

FIG. 1. Case 2: Small elastic fib& aggregate in the slthintimal region of t,he media which contains spindle-shaped and stellate cells. L, lumen. Epon embedding, t,oluidine blue. X 140. FIG. 2. Case 2: PAS-positive granules (arrows) are seen in many medial cells. Epon embedding, PAS. X 5G0. FIG. 3. Case 4: Condensed elastic fibers (arrow) separate the intima from the underlying medial lesion. The latter contains abundant ground substance and collagen (pale areas in photograph) and numerous small elastic fibrils. The architecture of the media is normal at N. L, lumen. Paraffin embedding, pentachrome I; X 9G. FIG. 4. Case 13: Numerous smooth muscle cells arranged in bundles. ParaffilL embedding, PTAI-I. X 5G0. no

AORTIC

COARCTATION

29

either from the lumen or from the adventit’ia, were often seen in the medial lesion. Pericapillary extravasation of red blood cells associated with iron-positive granules was only occasionally found. Eleciron

microscopic

obsewations

In newborn and young infants the cellular elements of the medial lesion lacked uniformity in their fine st,ructure. Some cells resembled fibroblasts. Most cellular elements, however, were smooth muscle cells which greatly varied in appearance (Figs. 5 and 6). Large areas of the cytoplasm were frequently occupied by aggregated glycogen granules (Fig. 5) which appeared to correspond to PAS-positive material seen by light microscopy in sections taken from adjacent areas. Many smooth muscle cells, with or without glycogen, contained large cisternas of the rough-surfaced endoplasmic reticulum (Fig. 6). All ‘(types” of smooth muscle cells were surrounded by a basement membrane, had numerous pinocytic vesicles along their cell membrane, and contained myofilaments which measured from 30-100 A in thickness. The int’ercellular spaces were loose, seemingly edematous, and contained numerous small granules (?glycogen), abundant amorphous basement membrane material, small elastic elements, and some collagen fibers (Figs. 5 and 6). In specimensfrom Cases3 and 4 (age of patients 8 months and 2% years, respectively) smooth muscle cells often contained membrane-bound inclusions (Figs. 7 and 8) but little glycogen. Many mitochondria appeared swollen (Fig. 7) and with other cytoplasmic organelles were frequently incorporated into the membranebound inclusions (Fig. S). The latter were, in places, in contact with the extracellular space (Figs. 7 and S, arrows). In addition, numerous microfibrils (Low, 1962) associated with small elastic elements and collagen, appeared in the extracellular space (Fig. 9). In aortas of young children, smooth muscle cells revealed further changes, while membrane-bound inclusions were seenwith decreasing frequency. Thus, numerous clusters of ribosome particles were observed in poorly defined, vacuolated areas which in places were bordered by patches of condensed myofilaments, the so-called fusiform densities. Moreover, myofilaments connected with such fusiform densities were also recognized in vacuolated areas (Fig. 10). In coarctations obtained from children older than 5 years, smooth muscle cells assumeda compact fibrillar cytoplasm, contained fewer ergastoplasmic membranes than smooth muscle cells of control aortas and were increasingly associated with abundant fibroelastic tissue (Fig. 11). The latter had a characteristic plexiform pattern due to a close association of collagen with elastic elements. In addition, the matrix of most elastic fibers contained numerous fenestrations filled with collagen (Figs. 11 and 12). This pattern of the fibroelastic tissue was not observed in normal aortas or in intimal thickenings. In areas of the medial lesion associatedwith excessive amounts of collagen, there were few microfibrils in the extracellular space and the feneskated elastic element’sappeared fragmented and disint#egrated (Fig. 13). Aggregation of numerous elastic unit’s (Haust et al., 1965) about atrophic smooth muscle cells was a characteristic feature of the medial lesion (Fig. 14). In contrast, similar cells in intimal thickenings showed a thick fibrillar basement membrane in association with collagen rat’her than with elastic element,s(Fig. 15).

FIG. 5. Case 2: Large aggregates of glycogen granules in a smooth muscle cell. gl, glycogen; ci, cisterna of the endoplasmic reticulum; mf, myofilaments; pv, pinocytic vesicles; bm, basement membrane; r, ribosome particles. Numerous small granules are noted in the extracellular space (arrows). Lead acetate. X 41,600. FIG. 6. Case 2: Dilated rough cisterna (ci) in smooth muscle cell. Small elastic fibrils (el) and amorphous basement membrane material are noted in the loose extracellular space. bm, basement membrane; n, nucleus. Lead acetate. X 22,000. 30

FIG. 7. Case 3: Swollen mitochondria (m), dilated rough cisternas (ci) and numerous cytoplasmic bodies (cb) in smooth muscle cells. Products of cellular degeneration are noted in the extracellular space (arrow). r, ribosomes; mf, myofilaments. Lead acetate. X 51,600. FIG. 8. Case 3: Degenerated portion of a smooth muscle cell. Altered mitochondria and other cytoplasmic elements are noted in the membrane-limited cytoplasmic body, which in places seems to be in contact with the extracellular space (arrow). mf, myofilaments; er, endoplasmic reticulum. Lead acetate; X 25,000. FIG. 9. Case 3: Numerous thin microfibrils associated with small elastic elements (el) and collagen are noted in the extracellular space. SM, smooth muscle cell; pv, pinocytic vesicles. Lead acetate; X 48,000. 31

FIG. 10. Case 4: Fusiform densit,ies (fd) Ijorder poorly defined vacuolatrd areas (v), whirh contain myofilaments and clusters of rihosome parlicles (r). F, fat; er, endoplasmic retirldum. Lead acetate; X 50,000. FIG. 11. Case 13: Portion of a well differentiated smooth muscle cell (WI). Numerolls fcnestrated elastic fibers (el) and some collagen (co) are noted in the estracelllllar space. Urallyl acetate; X 23,000. FIG. 12. Case 13: At high magnification collagen fibers are seen in fellestrations of the elastica (arrows). co, collagen. Uranyl acetate. X G2,OOO. 32

FIG. 13. Case 16: Large acellular areas of the media contain abundant collagen (co) and some fragments of elastic tissue (el). Uranyl acetate. X 23,000. FIG. 14. Case 13: Smooth muscle cell (SM) with little fibrillar cytoplasm is surrounded by numerous small elastic elements. co, collagen; el, elastic fibrils. Uranyl acetate. X 23,600. FIG. 15. Case 16: Area from intimal thickening. Portion of a smooth muscle cell with scanty cytoplasm is surrounded by a fibrillar basement membrane (arrows). co, collagen; n, nucleus of smoot,h muscle cell. TJranvl acetate. X 56.000.

34

J.

U.

BALIS,

A.

S.

CHAN,

AND

P.

E.

CONEN

DISCUSSION Originally, Bonnet (1903) classified coarctation of the aorta into two groups, infantile and adult types. Subsequently various classifications have been introduced (Edwards et al., 1948; Mustard et al., 1955, DeBoer et al., 1961) which consider anatomical, clinical, hemodynamic and surgical data. From the histopathological viewpoint this congenital malformation may be defined as a medial myoproliferative lesion which is accompanied by progressive increase of fibroelastic tissue. The etiology of aortic coarctation is unknown. In agreement with observations previously made by Edwards and his associates (Edwards et al., 1948; Clagett et al., 1954) our findings strongly suggest that ductal tissue is not involved in the formation of the medial lesion. The latter is possibly related to an in utero injury of the aortic wall. Some experimental evidence may support this concept: the repair process of medial dissecting aneurysms, produced in utero in rat aortas with beta-aminopropionitrile, is also characterized by proliferation of fibroelastic tissue and smooth muscle cells (Balis et al., 1965). CelZuZar elements The medial lesion appears to develop with interstitial edema and proliferation of poorly differentiated smooth muscle cells and some fibroblasts. There are many papers dealing with the fine structure of smooth muscle cells (Pease and Paule, 1960; Keech, 1960; Karrer, 1961) and their role in human (Haust et al., 1960; Geer et al., 1961; Balis et al., 1964 and experimental (Parker, 1960; Buck, 1961, 1963; Thomas et al., 1963; Hartroft and Thomas, 1963; Scott et al., 1964) lesions of the aorta. In addition, the evolution of smooth muscle from the undifferentiated mesenchymal cells, “fibroblasts,” has been studied sequentially in chick embryo aortas (Karrer, 1960). There are no published morphological studies describing glycogen in smooth muscle cells of elastic arteries, although glycogen has been found in arterioles (BiavaJ963, Zamboni andwestin, 1964) andmuscular arteries (Schmidt and Hillenbrand, 1953). It appears from our observations, that in human aorta, glycogen accumulates in poorly differentiated smooth muscle cells (Fig. 6) and it is eliminated in later stages of their differentiation. Kirk (1963) reached a similar conclusion from biochemical studies on glycogen phosphorylase activity of human aortas and coronary arteries. These studies suggested reduction in glycogen phosphorylase activity with aging and arteriosclerosis. Among other tissues of mesenchymal origin, transitory glycogen also appears in developing fat cells (Napolitano, 1963). In contrast, a reverse process seems to occur in developing chondroblasts which contain less glycogen than mature chondrocytes (Godman and Porter, 1960). The significance of these findings is obscure. It is possibly not a mere coincidence, however, that little glycogen but increasing numbers of membrane limited cytoplasmic bodies (autophagic vacuoles) are observed in smooth muscle cells with advancing age. Autophagic vacuoles have been described in injured cells in many physiological and pathological conditions (Ashford and Porter, 1962; Hruban et al., 1963; Novikoff, 1963). They are believed to reflect focal cytoplasmic degradation (Hruban et al., 1963) as a response to injury or physiological stimuli.

AORTIC

COARCTATION

35

It has been long recognized that cellular autolysis is prominent in embryonal tissues and may play a morphogenetic role in developmental processes (Glticksmann, 1951). Moreover, accumulation of autophagic vacuoles has been observed in embryonal and postnatally different#iating cells (Behnke, 1963 ; Balis and Conen, 1964; Balis et aZ., 1964). It has been suggested (Ghicksmann, 1951; Ashford and Porter, 1962) that the breakdown products included in autophagic vacuoles are eventually utilized by the cells. In addition, residual material from these vacuoles is possibly released into the extracellular space (Figs. 8 and 9, arrows). A similar process has been suggested in developing alveolar epithelium (Balis and Conen, 1964). In a further stage of differentiation, smooth muscle cells reveal few autophagic vacuoles but many poorly defined vacuolated areas which contain thin myofilaments in association with aggregated ribosome particles. Such changes are also found in differentiating medial cells of human fetal aortas (Chan et al., 196.5) and suggest that myofilaments are formed at the level of ribosome particles. This interpretation is supported by the work of Price et al., (1964) who also described a close association of myofilaments with polyribosomes in regenerating skeletal muscle. Extracellular

elements

There is now a large body of evidence (Revel and Hay, 1963; Porter, 1964; Goldberg and Green, 1964) which suggests that the rough endoplasmic reticulum of fibroblasts (Movat and Fernando, 1962) and other mesenchymal cells (Godman and Porter, 1960; Karrer, 1960; Cameron, 1961) is involved in the synthesis and segregation of collagen and other extracellular elements. In addition, morphological and biochemical studies of established mouse fibroblast lines (Goldberg and Green, 1964) have suggested that collagen accumulates in cultures only in the postproliferative phase (stationary phase), when the cells have demonst’rated a well-developed endoplasmic reticulum. These considerations may also apply to arterial smooth muscle. The latter resemble fibroblasts during embryonal life (Karrer, 1960). In aortic coarctations, fibroblast-like cells and smooth muscle cells with few but large rough cisternas are commonly observed in early stages of the medial myoproliferative lesion. In contrast, smooth muscle cells, like fibrocytes, show little cytoplasm and scanty mitochondria and ergastoplasmic membranes in advanced stages of differentiation. It may be assumed, t’herefore, that a correlation exists between the sequence of events observed in the extracellular spaces of the medial lesion and the various phases of cytodifferentiation. Thus, when cells are rich in endoplasmic reticulum (early stages of the lesion), thin microfibrils (Low, 1962) and amorphous basement membrane material predominate in extracellular spaces. These elements are possible precursors of the fibroelastic tissue since they are not very prominent in later stages of the lesion when cells are more differentiated and fibroelast,ic tissue increasingly accumulates in extracellular spaces. In older lesions, fraying and fragmentation of the elastic fibers seems to coincide with regressive changes in the smooth muscle cells. Similar alterations have been described by Karrer (1961) in aortas of aging mice. This suggests that smooth muscle cells play an important role in the integrity of the elastic tissue.

36

J.

U.

BALIS,

A.

S.

CHAN,

AND

P.

E.

CONEN

SUMMARY The

congenital medial lesion of aortic coarctation was studied by light and electron microsIn aortas of young infants prominent morphological features of this lesion were interstitial edema, increased ground substance and proliferation of poorly differentiated smooth muscle cells. The latter contained large aggregates of glycogen granules and frequent profiles of dilated endoplasmic reticulum. Characteristic changes were observed in aortic coarctation with age. Thus, there was progressive accumulation of fibroelastic tissue in the extracellular space. Moreover, autophagic vacuoles, suggestive of focal cytoplasmic degradation, appeared in smooth muscle cells, which also revealed a gradual decrease in their glycogen content. Subsequently, there were frequent vacuolated areas in the cytoplasm which contained thin myofilaments associated with numerous clusters of ribosome particles. Elastic elements often showed evidence of degeneration in coarctations obtained from children older than 5 years. At this time, smooth muscle cells appeared well differentiated and contained scanty ergastoplasmic membranes. These observations suggest that the morphogenesis of arterial fibroelastosis is interrelated with various phases of cytodifferentiation. It is furt,her suggested that aging of the arterial tissue is accelerated in areas of structural disorganization. copy.

ACKNOWLEDGMENTS The authors wish to thank Drs. W. A. Mustard and G. A. Trusler for providing the surgical specimens and clinical details, and Drs. W. Firor and M. Sroudji for their cooperation. The technical assistance of Mrs. Jacqueline Patterson and Miss Rollande Tremblay and the secretarial assistance of Mrs. Dorothy Fade are gratefully acknowledged. REFERENCES ASHFORD, T. P., and PORTER, K. R. (1962). Cytoplasmic components in hepatic cell lysosomes. J. Cell Biol. 12, 198-202. BALIS, J. U., CHAN, A. S., and CONEN, P. E. (1964). Electron microscopic study of the developing human liver. T. Gastroent. 7b, 133-148. BALE, J. U., CHAN, A. S., and CONEN, P. E. (1966). Lathyrogenic injury to foetal rat aortas and post-natal repair. E~ptl. Mol. Pathol. 5, 39-12. BALIS, J. U., and CONEN, P. E. (1964). The role of alveolar inclusion bodies in the developing lung. Lab. Invest. 13, 1215-1229. BALIS, J. U., HAUST, M. D., and MORE, R. H. (1964). Electron microscopic studies in human atherosclerosis. Cellular elements in aortic fatty streaks. Exptl. Mol. Pathol. 3, 511-525. BEHNKE, 0. (1963). Demonstration of acid phosphatase-containing granules and cytoplasmic bodies in the epithelium of foetal rat duodenum during certain stages of differentiation. J. Cell Biol. 18, 251-265. BIAVA, C. (1963). Identification and structural forms of human particulate glycogen. Lab. Invest. 12, 1179-1197. BONNET, L. M. (1903). Sur la lesion dite stenose congenitale de l’orte, dans la region de l’isthme. Rev. Med. Paris 23, 108-126. BUCK, R. C. (1961). Intimal thickening after ligature of arteries. An electron microscopic study. Circulation Res. 9, 418426. BUCK, R. C. (1963). Histogenesis and morphology of arterial tissue. In “Atherosclerosis and Its Origin” (M. Sandler and G. H. Bourne, eds.), pp. l-36. Academic Press, New York. CAMERON, D. A. (1961). The fine structure of osteoblasts in the metaphysis of the tibia of the young rat. J. Biophys. Biochem. Cytol. 9, 583-595. CAULFIELD, J. B. (1957). Effects of varying the vehicle of 0~04 in tissue fixation. J. Biophys. Biochem. Cytol. 3, 827-830. CHAN, A. S., BALIS, J. U., and CONEN, P. E. (1965). Maturation of smooth muscle cells in developing human aorta. Amt. Record 151, 334. (Abstract.)

AORTIC

COARCTATION

37

CLAGETT, 0. T., KIRKLIN, J. W., and EDWARDS, J. E. (1954). Anat’omic variations and pat.hologic changes in coarctation of the aorta. Surg. Gynecol. Obstet. 98, 103-114. DEBOER, A., GRANA, L., POTTS, W. J., and LEV, M. (1961). Coarctation of aorta. Arch. surg. 82, 80-812. EDWARDS, J. E., CHRISTENSEN, N. A., CLAGETT, 0. T., and MCDONALD, J. R. (1948). Pathologic considerations in coarctation of the aorta. Proc. Mayo CZin. 23, 324-332. GEER, J. C., MCGILL, H. C., and STRONG, J. P. (1961). The fine structure of human atherosclerotic lesions. Am. J. Pathol. 38, 263-287. GLASS, I. H., MUSTARD, W. T., and KEITH, J. D. (1960). Coarctation of the aorta in infants. Pediatrics 26, 109-121. GL~~CKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. GODMAN, G. C., and PORTER, K. R. (1960). Chondrogenesis, studied with the electron microscope. J. Biophys. Biochem. Cytol. 8, 719-760. GOLDBERG, B., and GREEN, H. (1964). An analysis of collagen secretion by established mouse fibroblast lines. J. Cell Bid. 22, 227-258. HARTROFT, W. S., and THOMAS, W. A. (1963). Induction of experimental atherosclerosis in various animals. In “Atherosclerosis and Its Origin” (M. Sandler and G. H. Bourne, eds.), pp. 439-456. HAUST, M. I)., MORE, It. H., BENCOSME, 8. A., and BALIS, J. U. (1965). Elastogenesis in human aorta: An electron microscopic study. Ezptl. Mol. Pathol. 4, W-524. HAUST, M. D., MORE, R. H., and MOVAT, H. Z. (1960). The role of smooth muscle cells in the fibrogenesis of arteriosclerosis. Am. J. Puthol. 37, 377-389. HRUBAN, Z., SPARGO, B., SX~IFT, H., WISSLER, R. W., and KLEINFELD, It. G. (1963). Focal cytoplasmic degradation. Am. J. Pathol. 42,657-683. KARRER, H. E. (1960). Electron microscope study of developing chick embryo aorta. J. Ultrastruct. Res. 4, 42G454. KARRER, H. E. (19Gl). An electron microscopestudy of the aorta in young and in aging mice. J. Ultrastruct. Res. 5, l-27. KEECH, M. K. (1960). Electron microscope study of the normal rat aorta. J. Biophys. Biothem. Cytol. 7, 533-538. KIRK, J. E. (1963). Intermediary metabolism of human arterial tissue and its changes with age and atherosclerosis. In “Atherosclerosis and Its Origin” (M. Sandler and G. H. Bourne, eds.), pp. 67-117. Academic Press, New York. Low, F. N. (1962). Microfibrils: Fine filamentous components of the tissue space. Anal. Record 142, 131-137. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. &o&em. Cytol. 9, 409414. MILLONIG, G. A. (1961). A modified procedure for lead staining of thin sections. J. Biophys. Biochem. Cytol. 11, 736-739. Mov‘yr, H. Z. (1955). Demonstration of all connective t.issue elements in a single section. Arch. Path&. 60, 289-295. MOVAT, H. Z., and FERNANDO, N. V. (1962). The fine structure of connective tissue. I. The fibroblast. Exptl. Mol. Pathol. 1, 509-534. MusraRD, W. T., ROLE, R. D., KEITH, J. D., and SIREK, A. (1955). Coarctation of the aorta with special reference to the first year of life. Ann. Surg. 141, 429436. NAPOLITANO, L. (1963). The differentiation of white adipose cells. An electron microscope study. J. Cell Bid. 18, 663-679. NOVIKOFF, A. B. (1963). Lysosomes in the physiology and pathology of cells: Contributions of staining methods. In “Lysosomes” (Ciba Foundation Symposium), (A. V. 8. de Reuck, and M. P. Cameron, eds.) pp. 36-77. Churchill, London. PARKER, F. (1960). An electron microscopic study of experimental atherosclerosis. Am. J. Pathol. 36, 19-53. PEASE, D. C., and PXCJLE, W. J. (1960). Electron microscopy of elastic arteries. The thoracic aorta of the rat. J. Ultrastrucl. Res. 3,469483.

38

J.

U.

BALIS,

A.

S.

CHAN,

AND

P.

E.

CONEN

(1964). Cell fine structure and biosynthesis of intercellular macromolecules. PORTER, K. R. Biophys. J., Suppl., 4, 167-201. PRICE, H. M., HOWES, E. L., and BLUMBERG, J. M. (1964). Ultrastructural alterations in skeletal muscle fibers injured by cold. II. Cells of the sarcolemmal tube: Observations on “discontinuous” regeneration and myofibril formation. Lab. Invest. 13, 1279-1302. REIFENSTEIN, G. H., LEVINS, S. A., and GROSS, R. E. (1947). Coarctation of the aorta. A review of 104 autopsied cases of the “adult type,” 2 years of age or older. Am. Heart J. 33, 146-168. REVEL, J. P., and HAY, E. D. (1963). A n autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage. 2. ZeZZforsch. 61, 116-144. (1953). Untersuchungen iiber Arteriosklerose und SCHMIDT, C. G., and HILLENBRAND, H. J. Endangitis Obliterans; der Glykogenehalt der Arterien bei Arteriosklerose und Endangitis Obliterans. 2. Ges. Expfl. Med. 120, 685-695. SCOTT,R. F., MORRISON, E. S., THOMAS, W. A., JONES, R. and NAM, S. C. (1964). Short term feeding of unsaturated versus saturated fat in the production of atherosclerosis in the rat. Exptl. Mol. Pathol. 3, 421443. THOMAS, W. A., JONES, R., SCOTT,R. F., MORRISON, E., GOODALE, F., and IMAI, H. (1963). Production of early atherosclerotic lesions in rats characterized by proliferation of “modified smooth muscle cells.” Exptl. Mol. Pathol. Suppl. 1, 40-60. ZAMBONI, L., and WESTIN, B. (1964). The ultrastructure of the human fetal spleen. I. One type of mesenchymal cell in the early stage of development of the spleen. J. Ultrastruct. Res. ll,,

469493.