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Heterogeneity of smooth muscle cells in embryonic human aorta
Tissue & Cell, 1995 27 (1) 31-38 © 1995 Pearson Professional Ltd.
Heterogeneity of smooth muscle cells in embryonic human aorta A. A. Mironovt, M. D. Rekhter:~, V. A. Kolpakov:l:, E. R. Andreeva*, R. S. Polishchuk, S. I. Bannykhtt, S. V. Filippov, L. P. Peretjatko, L. V. Kulida, A. N. Orekhov*
Abstract. Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for 0~-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.
Keywords: Cellularcomposition, heterogeneity,humanembryo aorta, morphology,smooth musclecells
Introduction The subendothelial intima of adult human aorta contains smooth muscle cells (SMC) and blood born cells as monocytes/macrophages, lymphocytes etc. (Gown et al., 1986). Smooth muscle cells play a key role in atherogenesis (Campbell and Campbell, 1989; Ross, 1986; Schwartz et al., 1986). Some investigators believe that SMC located in the intima are normal intimal components, while others claim that intimal thickening Ivanovo State Medical Institute, Ivanovo, Russia. *Institute of Experimental Cardiology, Moscow, Russia. Received 8 June 1994 Accepted 23 August 1994 Correspondence to; Elena R. Andreeva, Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Street, 15a, 121552 Moscow~ Russia. Present address: t Consorzio Mario Negri Sud, Italy. University of Michigan, Ann-Arbor, MI, USA. t t University of California, San Diego, CA, USA.
and emergence of SMC in the subendothelial layer represent a step in the development of atherosclerosis, and are associated with SMC migration from the media (Campbell and Campbell, 1989; Schwartz et al., 1986). The population of intimal SMC is polymorphous; in addition to typical spindle shaped cells, stellate cells that differ from typical SMC by less developed contractile structures and better developed synthetic apparatus have also been found (Orekhov et al., 1986). In an atherosclerotic lesion, numerous SMC resemble poorly differentiated embryonic SMC in the ultrastructure, protein composition of the cytoskeleton, expression of surface antigens, etc. (Campbell, 1989; Glukhova et al., 1989; Nikkari et al., 1988; Schwartz et al., 1986). The investigation of human aortic intimal cells in prenatal ontogenesis may resolve the questions of the intima-media interaction in developing aorta and provide a better understanding of the cellular aspects in normal physiology and pathology of the vascular wall. In this study we have examined the cellular composi31
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tion of the subendothelial intima and media of human embryonic aorta.
Materials and methods The study was performed on 5- to 12-week-old (n = 14) and 18- to 28-week-old (n= 13) human embryos. The material was collected after artificial abortions performed at the Ivanovo Gynecology Clinic. Descending aortas were incised within 1 h after the operation. The specimens were washed with phosphatebuffered saline (pH 7.6) to remove blood and fixed with 2.5% glutaraldehyde in Medium 199 for 24 h.
Alcohol-alkaline dissociation In order to examine cell shape, aorta specimens were subjected to alcohol-alkaline dissociation (Orekhov et al., 1986). Before the treatment, a portion of the aorta was excised to control the accuracy of mechanical dissection and to carry out the electron microscopy studies. Dissociation of tissue was performed by incubation of aorta samples in a mixture of equal volumes of 30% KOH and 96% ethanol at 37°C with periodical shaking until complete dissolution of the tissue. An aliquot of the incubation medium was withdrawn each 30 rain and examined by phase-contrast microscopy; the number of cells of various shapes was counted (at least 200 cells were analyzed). Then the sample was immersed in a fresh portion of alcohol-alkaline mixture. Light and transmitting electron microscopy (TEM) Cross-sectional circles (1-2 mm thick) were cut from aorta, washed in Medium 199 (Gibco, Paisley, UK), post-fixed in 1% OsO4 for 1 h, washed with distilled water, treated with 1% tannic acid, washed, dehydrated in graded alcohols and absolute acetone, and embedded in Araldite M. Sections perpendicular to the long vessel axis were cut in a LKB-III microtome, stained with methylene blue and examined in a light microscope. Then ultrathin sections were cut from the area of interest, stained with uranyl acetate and lead citrate using conventional technique, examined and photographed in a transmitting electron microscope. The volume density of microfilaments was determined according to Campbell and Campbell (1985). Scanning electron microscopy (SEM) To study three-dimensional cytoarchitecture of embryonic aortic wall, segments of aorta were incubated in a KOH-ethanol mixture as described previously. The incu-
bation medium was examined by phase-contrast microscopy at 10-min intervals. To visualize subendothelial structures in the aortas of 18 to 24 week fetuses, 1-cm long segments were turned inside out so that the endothelium was located outside. The segment ends were clamped with microsurgical forceps, and the obtained tube was incubated in a KOHethanol (96%) mixture shaking at 37°C. To study the three-dimensional composition of the media, aortic segments were opened with scissors, mechanically separated and dissociated as described previously. After dissociation, tissue samples were washed with distilled water (vigorous shaking 3 times for 10rain) and processed for SEM. The postfixation and dehydration procedures were the same as for TEM. Dehydrated preparations were critical-point-dried in an HCP-2 (Hitachi Ltd, Kioto, Japan), coated with platinum in a JFC-1100 apparatus (Hitachi) and examined under a Hitachi S-570 electron microscope at an accelerating voltage of 20 kV.
Immunocytochemistry Aortic samples were fixed in Methyl-carnoy's fixative (methanol : chloroform: glacial acetic acid 6 : 3 : 1), embedded in Paraplast (Polysciences Inc., Warrington, USA) and 5 gm sections were prepared. The following antigens were detected using monoclonal antibodies: muscle ~-actin HHF-35, macrophage HAM-56 antigen (the antibodies were a generous gift of Dr A. Gown, University of Washington, Seattle, USA), myosin (the antibody was a generous gift of Dr M. G. Frid, Institute of Experimental Cardiology, Russia). Macrophage antigens were detected using Leu M3 monoclonal antibody (Becton-Dickinson, San-Jose, USA) and antimacrophage monoclonal antibody (Amersham International plc., Amersham, UK). Bound antibodies were visualized using a Vectastain kit (Vector Laboratories, Burlingame, USA).
Results Structure of aortic SMC from 5- to 12-week-old embryos Using light microscopy, the intima was not discerned under the endothelial monolayer in 5- to 12-week-old embryonic aorta. However, we found three cellular sublayers, oriented consequently from the endothelium to the outer side of the vessel. There were no anatomic borders of the elastic-membrane type between these sublayers.
Fig. 1 Structure of 5 to 12-week-old embryonic aorta. (a) Transverse semithin section, tt round cell. Methylene blue, x 550. (b) Longitudinal semithin section, t - round cell, ~ - elongated cell, • mitosis. Methylene blue, x 550. (c) Ultrastructure of a round cell. t ~ - microfilament bundle, tt - cistern o f R E R . TEM, x 27 000. • - tight junction. (d) Inner sublayer. R o u n d SMC. SEM, x 1250. (e) Intermediate sublayer. Elongated SMC, connected with cytoplasm bridge, (*). SEM, x 2200. (f) Alcohol-alkaline dissociation. * round-shaped cells. Phase contrast, x 1150. (g) Immunocytochemical identification of embryonic aortic cells. Smooth muscle c~-actin, for detection ABC-method (avidin-biotincomplex) was used, x 300.
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The major differences between these sublayers consisted of cell shape and packing. The inner sublayer adjacent to the endothelium contained 3 to 4 rows of round or ovoid cells. It should be mentioned that these cells were round on sections regardless of the plane in which they were cut (see Fig. la and lb). This indicates that these cells are almost spherical. The spherical shaped cells often formed clusters separated from each other by thin layers of an extracellular matrix. The cells of the inner sublayer had a well defined rough endoplasmic reticulum, numerous free ribosomes, great number of mitochondria and well developed Golgi apparatus. Microfilament bundles with dense bodies were not well developed. Occasional microvesicles were seen at the plasmalemma. The basal lamina was weakly developed (Fig. lc). Collagen fibers and small elastin lumps were seen between cells. The intermediate sublayer contained 3 or 4 cell rows. The cells were predominantly spindle shaped, evenly distributed and densely packed. There were mitotic cells at the borderline between the inner and intermediate sublayers (Fig. lb). Ultrastructurally, the intermediate layer cells were similar to the inner layer cells, but contained more microfilament bundles with dense bodies and microvesMes, which sometimes were arranged in rows and groups at the cytoplasm periphery. These sublayer cells resembled differentiated SMC. In the intermediate sublayer, round and ovoid cells were found by SEM in the area close to the lumen; these cells formed a multilayer stratum (Fig. ld) and were arranged in knot- and band-like structures. These structures may correspond to the clusters formed by the round cells found on the sections. Generally, these cells were in contact with their side surfaces with thin connective tissue layers between them. The surface of majority of the cells was covered with microvilli, sometimes with undulations, though cells with smooth surface were also seen. In the outer sublayer, S E M revealed a multilayer stratum of spindle shaped cells with cell-to-cell contacts or contact by the side surfaces either directly or with the aid of cytoplasmic bridges (Fig. le). The surface of these cells was covered with a small number of microvilli. The connective tissue layers were seen between the cells. The cytoplasm of these cells was filled predominantly by the rough endoplasmic reticulum, there were occasional microfilament bundles without dense bodies. There was no basal lamina. Ultrastructurally, these cells resembled fibroblasts or poorly differentiated mesenchymal cells. During alcohol-alkaline dissociation from the outer sublayer, cells of various shapes (spindle, stellate and
round) first appear in the suspension, then only spheric and polygonal cells appear (Fig. l f). This confirms the microscopy data and allows us to conclude that the aorta of a 5 to 12 week-old embryos contains spheric cells located beneath the endothelium and elongated cells located at the outer sublayers.
Immunocytochemieal analysis of early embryo aortas Aortas of 5-to-12-week-old embryos were examined with antibodies against muscle ~-actin, myosin and macrophage antigens. All cells in early embryo aorta positively immunostained with antimuscle ~-actin (Fig. lg) and antimyosin antibodies (not shown). Immunostaining with antimacrophagal antibodies was negative. Aortic smooth muscle cells of 18- to 28-week-old embryos Intima. The intima was readily distinguished due to a well developed internal elastic lamina (IEL) that serves as a morphologic borderline between intima and media. Cells were extremely rare between the endothelial monolayer and the IEL. Occasional separate cells or processes of medial cells penetrating the pores of IEL were seen in some areas of the aorta. Cells were more frequent in the subendothelial space of 25- to 28-week-old embryos (Fig. 2a). Generally, they formed groups consisting of 3 to 5 cells arranged in one row or occasionally in two rows. Aorta of 28-week-old embryos contained the greatest amount of subendothelial cells. Subendothelial cells were surrounded by an interrupted basal lamina; their cytoplasm contained numerous elements of the rough endoplasmic reticulum and small number of micro filaments (Fig. 2b). Some cells displayed considerable amounts of microtubules. Subendothelial cells often contacted with the processes of medial SMC and endothelial cells. The subendothelium contained both single and clustered cells. Clusterization was prevalent in the aortas of 25- to 28-week-old embryos. Within clusters, cells contacted with each other by their processes, forming a loose network (Fig. 2c). Cells of various shapes (stellate, Y- and spindle-like, elongated) were present in the intima. There were occasional separate round cells with a constricting ring in the central part (Fig. 2d). The major proportion of subendothelial cells had processes many of which were oriented to the endothelium (Fig. 2e). Free processes of stellate cells were often terminated by a knob-like widening or flattened triangle lamelloplasm. There were rows and 'fields' of roundish shallow craters on cell surface; by shape and size, these craters were similar to those revealed by the technique (Severs, 1988). In addition, the surface of
Fig. 2 Structureof 18 to 28-week-oldembryonicaorta. (a) Semithinsection, t - subendothelialcell, methyleneblue, x 450. (b) Ultrastructure of SMC (*). tt - basal membrane. ~ - RER. TEM, x 5500. (c) Cluster of stellate SMC (,). SEM, x 1250.(d) Round cellwith constrictingring (t). SEM, x 2000. (e) SMC surroundedby elastin (t), * - lumen. TEM, x 14000. (f) SMC contactingwith EC with the aid of processes(t), • lumen. TEM, x 6500.
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Table 1 Content of cells of various shape of embryonic human aorta
(%_+ S.E.M.) Embryo age
Spindle-shaped
Y-shaped
Stellate
Spherical
5-12 week 28-20 week
44.7_+1.8 75.0_+2.1"
11.8_+1.2 6.3_+1.2 37.2_+1.7 15.8_+1.8" 16.0_+1.6' 0
* Significantly different values in vertical columns (p < 0.01 ). Fixed aortic specimens (a media-intima preparation) were dissociated in an alcohol-alkaline mixture as described in Materials and Methods. Cells were counted in suspension using a hemocytometer
intimal cells had small transverse creases, microvilli and blebs. Lumps of elastin, which sometimes formed a structure resembling an additional elastic lamina, were seen in all cases in the space between subendothelial cells and endothelium (with the exception of the direct contact zones). Such a 'lamina' outlines the borders of subendothelial cells (Fig. 2e). There were collagen fiber bundles in the interstitium. Table 1 shows the proportion of ceils of various shape in embryonic aortas. Spindle shaped and round cells predominated in the aortas of 5- to 12-week-old embryos, while in 18- to 28-week-old embryonic aortas round cells disappear and elongated cells predominated. Stellate and Y-shaped cells were less frequent.
Media. In the aorta of 18- to 28-week-old embryos, medial SMC formed several rows which were separated from each other by an elastic lamina. Cells were loosely and randomly packed in the first 1 to 2 rows near the elastic lamina, while in the deeper layers they were densely packed. Intercellular contacts were quite frequent. There was crude collagen matrix around these cells. Media contained cells whose ultrastructure was similar to that of intimal cells. There were also cells with well developed basal lamina. The cytoplasm of these cells contained thick microfilament bundles (Fig. 3a). Microvesicle rows were seen predominantly at the cytoplasm periphery. Alcohol-alkaline dissociation of the media yielded a cell suspension containing spindle, Y and stellate-shaped cells. There were no round cells in this suspension (Fig. 3b). The number of spindle and stellate-shaped cells in aortas of 18- to 28-week-old embryos was significantly greater compared with aortas of 5- to 12-week-old embryos. The medial layer adjacent to the IEL predominantly contained cells with processes and smooth surface covered with occasional cavellas. The processes of the majority of these cells contacted with each other. However, blind cellular extensions were essentially frequent (Fig. 3c). In the deeper (intermediate and outer) layers of media,
cell packing, shape and microrelief differed considerably from those described previously. Almost all cells were spindle shaped. There were long longitudinal creases and rows of round pits on the cell surface. Cells were densely packed and contacted by their side surfaces, creases or cell surfaces overlaying each other in a tilelike manner. Interestingly, several neighboring cells formed complexes (bundles) within which they were arranged in parallel rows. The complexes were oriented at an angle to each other (Fig. 3d).
Imunocytochemical analysis of 18-28-weeks-old embryo aortas Intimal and medial cells were intensely stained with antiactin (Fig. 3e) and anti-myosin (not illustrated) antibodies in the aortas 18- to 28-week-old embryos. Occasionally cells in the adventitia and adjacent lymph nodes were reactive with anti-macrophage antibodies. Morphologically, they were typical monocytes (not illustrated). Cells of embryonic aorta contained no macrophage antigens.
Discussion In this study, we have demonstrated that embryonic human aorta contains SMC differing in their ultrastructure, shape, packing and surface microrelief. Previously we reported on the heterogeneity on the intimal cell population in adult human aorta (Orekhov et al., 1986). Both embryonic and adult aortas contain elongated and stellate SMC. This study shows that the morphological basement of this phenomenon is laid at the 18- to 28-week-old of embryogenesis. Cells located in different sublayers of embryonic aortic media have varied shape and form variety of contacts with each other. Interestingly, SMC of the media closest to lumen have free (noncontacting) processes which can penetrate into the intima. This may be either associated with the aortic intima formation, or may reflect the processes of cell migration from the intima to the media. It is known that in human aorta, intimal cells appear in the early prenatal period (Fruntash, 1982; Kamenskaya, 1959). Their nature and origin have not been investigated. Intimal cells are directly connected with medial SMC via the internal elastic membrane. It may be suggested that the cells appear in the intima as a result of cell migration from the media. In embryonic intima we found separated or clustered cells. We assumed that one cell may migrate to the intima from the media, start dividing and form a cluster. As we have shown in present work all subendothelial cells of embryonic aorta contain SMC antigens.
Fig. 3 Structure of media in 18-28-week-old embryonic aorta. (a) Ultrastructure of SMC. t - microfilament bundle. TEM, x 23 000. (b) Cell suspension. Alcohol-alkaline dissociation, x 400. (c) SMC with blind processes, (t~). SEM, x 2000. (d) SMC forming strata in the inner layer of the media (*), oriented at an angle. SEM, x 400. (e) Immunocytochemical identification of medial aortic cells, smooth muscle ~-actin, for detection ABC-method (avidin-biotin-complex) was used, x 200.
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Irrespective of this fact, predominantly two cell populations consisting of spherical and elongated cells were isolated from human aortas at early stages of embryogenesis. Spherical and elongated cells differed ultrastructurally; the former were poorly differentiated and the latter had the major characteristics of differentiated SMC. The two cell populations differed in the character of cell packing. Spherical cells were densely packed, the connective tissue between them was practically absent, while elongated cells were separated from each other by defined layers of connective tissue. Two cell types were also found in the aortas of 18to 28-week-old embryos. Occasionally, poorly differentiated stellate cells were located in close proximity to the endothelium. The major bulk of medial cell population consisted of spindle-shaped SMC. Intimal-like
cells with processes were suited only in the innermost part of media near the IEL. Similar location of spheroid cells of early embryo, cells with processes of relatively late embryo and subendothelial stellate cells in adult human aorta offers us to propose, that spheroid cells of early embryo m a y be precursor cells for cells with processes and stellate cells, which appear on relatively late stages of embryogenesis. ACKNOWLEDGEMENTS We thank Mr Andrey Fesenko, Dr Igor Sobenin (Institute of Experimental Cardiology, National Cardiology Research Center, Moscow, Russia) and M r Suk Yoon (University of Michigan, Ann Arbor, MI, USA) for their help in the preparation of this manuscript.
REFERENCES Andreeva, E.R., Rekhter, M.D., Romanov, Yu.A., Antonova, G.M., Antonov, A.S., Mironov, A,A., Orekhov, A.N. 1992. Stellate cells of aortic intima: II. Arborization of intimal cells in culture. Tissue Cell, 24, 697-704. Campbell, G.R. and Campbell J.H. 1985. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp. Mol. Pathol., 42, 139-162. Campbell, J.H. and Campbell, G.R. 1989. Biology of the vessel wall and atherosclerosis. Clin. Exp. Hypertens. A., 11,901-913. Fruntash, N.M. 1982. Biomorphogenesisof human aorta. Schtiinza, Kischinev. (Russ). Glukhova, M.A., Frid, M.G., Shekhonin, B.V., Vasilievskaya,T.D., Grunvald, J., Saginati, M., Koteliansky, V.E. 1989. Expression of extra domain of fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J. Cell Biol., 109, 357-366. Gown, A.M., Tsukada, T. and Ross, R. 1986. Human atherosderosis. II. Immunocytochemicalanalysis of the cellular composition of human atherosclerotic lesions. Am. J. Pathol., 125, 191-207.
Kamenskaya, N.L. 1959. Materials on human aorta histogenesis. Arch. Anat. Hist. Embr., 36, 61-66. Nikkari, S.J., Rantala, I., Pystynen, P., Nikkari, T. 1988. Characterization of the phenotype of smooth muscle cells in human fetal aorta on the basis of ultrastructure, immunofluorescence, and the composition of cytoskeletal and cytocontractile proteins. Atherosclerosis, 74, 33-40. Orekhov, A.N., Krushinsky, A.V., Andreeva, E.R., Repin, V.S., Smirnov, V.N. 1986. Adult human aortic cells in primary culture: heterogeneity in shape. Heart Vessels, 2, 193-201. Rekhter, M.D., Andreeva, E.R., Mironov, A.A. and Orekhov, A.N. 1991. Three-dimensionalcytoarchitecture of normal and atherosclerotic intima of human aorta. Am. J. Pathol., 138, 569-580. Ross, R. 1986. The pathogenesis of atherosclerosis - an update. N. Engl. J. Med., 314, 488-450. Schwartz, S.M., Campbell, G.R., Campbell, J.H. 1986. Replication of smooth muscle cells in vascular disease. Circ. Res., 58, 427-444.
Heterogeneity of smooth muscle cells in embryonic human aorta A. A. Mironovt, M. D. Rekhter:~, V. A. Kolpakov:l:, E. R. Andreeva*, R. S. Polishchuk, S. I. Bannykhtt, S. V. Filippov, L. P. Peretjatko, L. V. Kulida, A. N. Orekhov*
Abstract. Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for 0~-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.
Keywords: Cellularcomposition, heterogeneity,humanembryo aorta, morphology,smooth musclecells
Introduction The subendothelial intima of adult human aorta contains smooth muscle cells (SMC) and blood born cells as monocytes/macrophages, lymphocytes etc. (Gown et al., 1986). Smooth muscle cells play a key role in atherogenesis (Campbell and Campbell, 1989; Ross, 1986; Schwartz et al., 1986). Some investigators believe that SMC located in the intima are normal intimal components, while others claim that intimal thickening Ivanovo State Medical Institute, Ivanovo, Russia. *Institute of Experimental Cardiology, Moscow, Russia. Received 8 June 1994 Accepted 23 August 1994 Correspondence to; Elena R. Andreeva, Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Street, 15a, 121552 Moscow~ Russia. Present address: t Consorzio Mario Negri Sud, Italy. University of Michigan, Ann-Arbor, MI, USA. t t University of California, San Diego, CA, USA.
and emergence of SMC in the subendothelial layer represent a step in the development of atherosclerosis, and are associated with SMC migration from the media (Campbell and Campbell, 1989; Schwartz et al., 1986). The population of intimal SMC is polymorphous; in addition to typical spindle shaped cells, stellate cells that differ from typical SMC by less developed contractile structures and better developed synthetic apparatus have also been found (Orekhov et al., 1986). In an atherosclerotic lesion, numerous SMC resemble poorly differentiated embryonic SMC in the ultrastructure, protein composition of the cytoskeleton, expression of surface antigens, etc. (Campbell, 1989; Glukhova et al., 1989; Nikkari et al., 1988; Schwartz et al., 1986). The investigation of human aortic intimal cells in prenatal ontogenesis may resolve the questions of the intima-media interaction in developing aorta and provide a better understanding of the cellular aspects in normal physiology and pathology of the vascular wall. In this study we have examined the cellular composi31
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tion of the subendothelial intima and media of human embryonic aorta.
Materials and methods The study was performed on 5- to 12-week-old (n = 14) and 18- to 28-week-old (n= 13) human embryos. The material was collected after artificial abortions performed at the Ivanovo Gynecology Clinic. Descending aortas were incised within 1 h after the operation. The specimens were washed with phosphatebuffered saline (pH 7.6) to remove blood and fixed with 2.5% glutaraldehyde in Medium 199 for 24 h.
Alcohol-alkaline dissociation In order to examine cell shape, aorta specimens were subjected to alcohol-alkaline dissociation (Orekhov et al., 1986). Before the treatment, a portion of the aorta was excised to control the accuracy of mechanical dissection and to carry out the electron microscopy studies. Dissociation of tissue was performed by incubation of aorta samples in a mixture of equal volumes of 30% KOH and 96% ethanol at 37°C with periodical shaking until complete dissolution of the tissue. An aliquot of the incubation medium was withdrawn each 30 rain and examined by phase-contrast microscopy; the number of cells of various shapes was counted (at least 200 cells were analyzed). Then the sample was immersed in a fresh portion of alcohol-alkaline mixture. Light and transmitting electron microscopy (TEM) Cross-sectional circles (1-2 mm thick) were cut from aorta, washed in Medium 199 (Gibco, Paisley, UK), post-fixed in 1% OsO4 for 1 h, washed with distilled water, treated with 1% tannic acid, washed, dehydrated in graded alcohols and absolute acetone, and embedded in Araldite M. Sections perpendicular to the long vessel axis were cut in a LKB-III microtome, stained with methylene blue and examined in a light microscope. Then ultrathin sections were cut from the area of interest, stained with uranyl acetate and lead citrate using conventional technique, examined and photographed in a transmitting electron microscope. The volume density of microfilaments was determined according to Campbell and Campbell (1985). Scanning electron microscopy (SEM) To study three-dimensional cytoarchitecture of embryonic aortic wall, segments of aorta were incubated in a KOH-ethanol mixture as described previously. The incu-
bation medium was examined by phase-contrast microscopy at 10-min intervals. To visualize subendothelial structures in the aortas of 18 to 24 week fetuses, 1-cm long segments were turned inside out so that the endothelium was located outside. The segment ends were clamped with microsurgical forceps, and the obtained tube was incubated in a KOHethanol (96%) mixture shaking at 37°C. To study the three-dimensional composition of the media, aortic segments were opened with scissors, mechanically separated and dissociated as described previously. After dissociation, tissue samples were washed with distilled water (vigorous shaking 3 times for 10rain) and processed for SEM. The postfixation and dehydration procedures were the same as for TEM. Dehydrated preparations were critical-point-dried in an HCP-2 (Hitachi Ltd, Kioto, Japan), coated with platinum in a JFC-1100 apparatus (Hitachi) and examined under a Hitachi S-570 electron microscope at an accelerating voltage of 20 kV.
Immunocytochemistry Aortic samples were fixed in Methyl-carnoy's fixative (methanol : chloroform: glacial acetic acid 6 : 3 : 1), embedded in Paraplast (Polysciences Inc., Warrington, USA) and 5 gm sections were prepared. The following antigens were detected using monoclonal antibodies: muscle ~-actin HHF-35, macrophage HAM-56 antigen (the antibodies were a generous gift of Dr A. Gown, University of Washington, Seattle, USA), myosin (the antibody was a generous gift of Dr M. G. Frid, Institute of Experimental Cardiology, Russia). Macrophage antigens were detected using Leu M3 monoclonal antibody (Becton-Dickinson, San-Jose, USA) and antimacrophage monoclonal antibody (Amersham International plc., Amersham, UK). Bound antibodies were visualized using a Vectastain kit (Vector Laboratories, Burlingame, USA).
Results Structure of aortic SMC from 5- to 12-week-old embryos Using light microscopy, the intima was not discerned under the endothelial monolayer in 5- to 12-week-old embryonic aorta. However, we found three cellular sublayers, oriented consequently from the endothelium to the outer side of the vessel. There were no anatomic borders of the elastic-membrane type between these sublayers.
Fig. 1 Structure of 5 to 12-week-old embryonic aorta. (a) Transverse semithin section, tt round cell. Methylene blue, x 550. (b) Longitudinal semithin section, t - round cell, ~ - elongated cell, • mitosis. Methylene blue, x 550. (c) Ultrastructure of a round cell. t ~ - microfilament bundle, tt - cistern o f R E R . TEM, x 27 000. • - tight junction. (d) Inner sublayer. R o u n d SMC. SEM, x 1250. (e) Intermediate sublayer. Elongated SMC, connected with cytoplasm bridge, (*). SEM, x 2200. (f) Alcohol-alkaline dissociation. * round-shaped cells. Phase contrast, x 1150. (g) Immunocytochemical identification of embryonic aortic cells. Smooth muscle c~-actin, for detection ABC-method (avidin-biotincomplex) was used, x 300.
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The major differences between these sublayers consisted of cell shape and packing. The inner sublayer adjacent to the endothelium contained 3 to 4 rows of round or ovoid cells. It should be mentioned that these cells were round on sections regardless of the plane in which they were cut (see Fig. la and lb). This indicates that these cells are almost spherical. The spherical shaped cells often formed clusters separated from each other by thin layers of an extracellular matrix. The cells of the inner sublayer had a well defined rough endoplasmic reticulum, numerous free ribosomes, great number of mitochondria and well developed Golgi apparatus. Microfilament bundles with dense bodies were not well developed. Occasional microvesicles were seen at the plasmalemma. The basal lamina was weakly developed (Fig. lc). Collagen fibers and small elastin lumps were seen between cells. The intermediate sublayer contained 3 or 4 cell rows. The cells were predominantly spindle shaped, evenly distributed and densely packed. There were mitotic cells at the borderline between the inner and intermediate sublayers (Fig. lb). Ultrastructurally, the intermediate layer cells were similar to the inner layer cells, but contained more microfilament bundles with dense bodies and microvesMes, which sometimes were arranged in rows and groups at the cytoplasm periphery. These sublayer cells resembled differentiated SMC. In the intermediate sublayer, round and ovoid cells were found by SEM in the area close to the lumen; these cells formed a multilayer stratum (Fig. ld) and were arranged in knot- and band-like structures. These structures may correspond to the clusters formed by the round cells found on the sections. Generally, these cells were in contact with their side surfaces with thin connective tissue layers between them. The surface of majority of the cells was covered with microvilli, sometimes with undulations, though cells with smooth surface were also seen. In the outer sublayer, S E M revealed a multilayer stratum of spindle shaped cells with cell-to-cell contacts or contact by the side surfaces either directly or with the aid of cytoplasmic bridges (Fig. le). The surface of these cells was covered with a small number of microvilli. The connective tissue layers were seen between the cells. The cytoplasm of these cells was filled predominantly by the rough endoplasmic reticulum, there were occasional microfilament bundles without dense bodies. There was no basal lamina. Ultrastructurally, these cells resembled fibroblasts or poorly differentiated mesenchymal cells. During alcohol-alkaline dissociation from the outer sublayer, cells of various shapes (spindle, stellate and
round) first appear in the suspension, then only spheric and polygonal cells appear (Fig. l f). This confirms the microscopy data and allows us to conclude that the aorta of a 5 to 12 week-old embryos contains spheric cells located beneath the endothelium and elongated cells located at the outer sublayers.
Immunocytochemieal analysis of early embryo aortas Aortas of 5-to-12-week-old embryos were examined with antibodies against muscle ~-actin, myosin and macrophage antigens. All cells in early embryo aorta positively immunostained with antimuscle ~-actin (Fig. lg) and antimyosin antibodies (not shown). Immunostaining with antimacrophagal antibodies was negative. Aortic smooth muscle cells of 18- to 28-week-old embryos Intima. The intima was readily distinguished due to a well developed internal elastic lamina (IEL) that serves as a morphologic borderline between intima and media. Cells were extremely rare between the endothelial monolayer and the IEL. Occasional separate cells or processes of medial cells penetrating the pores of IEL were seen in some areas of the aorta. Cells were more frequent in the subendothelial space of 25- to 28-week-old embryos (Fig. 2a). Generally, they formed groups consisting of 3 to 5 cells arranged in one row or occasionally in two rows. Aorta of 28-week-old embryos contained the greatest amount of subendothelial cells. Subendothelial cells were surrounded by an interrupted basal lamina; their cytoplasm contained numerous elements of the rough endoplasmic reticulum and small number of micro filaments (Fig. 2b). Some cells displayed considerable amounts of microtubules. Subendothelial cells often contacted with the processes of medial SMC and endothelial cells. The subendothelium contained both single and clustered cells. Clusterization was prevalent in the aortas of 25- to 28-week-old embryos. Within clusters, cells contacted with each other by their processes, forming a loose network (Fig. 2c). Cells of various shapes (stellate, Y- and spindle-like, elongated) were present in the intima. There were occasional separate round cells with a constricting ring in the central part (Fig. 2d). The major proportion of subendothelial cells had processes many of which were oriented to the endothelium (Fig. 2e). Free processes of stellate cells were often terminated by a knob-like widening or flattened triangle lamelloplasm. There were rows and 'fields' of roundish shallow craters on cell surface; by shape and size, these craters were similar to those revealed by the technique (Severs, 1988). In addition, the surface of
Fig. 2 Structureof 18 to 28-week-oldembryonicaorta. (a) Semithinsection, t - subendothelialcell, methyleneblue, x 450. (b) Ultrastructure of SMC (*). tt - basal membrane. ~ - RER. TEM, x 5500. (c) Cluster of stellate SMC (,). SEM, x 1250.(d) Round cellwith constrictingring (t). SEM, x 2000. (e) SMC surroundedby elastin (t), * - lumen. TEM, x 14000. (f) SMC contactingwith EC with the aid of processes(t), • lumen. TEM, x 6500.
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Table 1 Content of cells of various shape of embryonic human aorta
(%_+ S.E.M.) Embryo age
Spindle-shaped
Y-shaped
Stellate
Spherical
5-12 week 28-20 week
44.7_+1.8 75.0_+2.1"
11.8_+1.2 6.3_+1.2 37.2_+1.7 15.8_+1.8" 16.0_+1.6' 0
* Significantly different values in vertical columns (p < 0.01 ). Fixed aortic specimens (a media-intima preparation) were dissociated in an alcohol-alkaline mixture as described in Materials and Methods. Cells were counted in suspension using a hemocytometer
intimal cells had small transverse creases, microvilli and blebs. Lumps of elastin, which sometimes formed a structure resembling an additional elastic lamina, were seen in all cases in the space between subendothelial cells and endothelium (with the exception of the direct contact zones). Such a 'lamina' outlines the borders of subendothelial cells (Fig. 2e). There were collagen fiber bundles in the interstitium. Table 1 shows the proportion of ceils of various shape in embryonic aortas. Spindle shaped and round cells predominated in the aortas of 5- to 12-week-old embryos, while in 18- to 28-week-old embryonic aortas round cells disappear and elongated cells predominated. Stellate and Y-shaped cells were less frequent.
Media. In the aorta of 18- to 28-week-old embryos, medial SMC formed several rows which were separated from each other by an elastic lamina. Cells were loosely and randomly packed in the first 1 to 2 rows near the elastic lamina, while in the deeper layers they were densely packed. Intercellular contacts were quite frequent. There was crude collagen matrix around these cells. Media contained cells whose ultrastructure was similar to that of intimal cells. There were also cells with well developed basal lamina. The cytoplasm of these cells contained thick microfilament bundles (Fig. 3a). Microvesicle rows were seen predominantly at the cytoplasm periphery. Alcohol-alkaline dissociation of the media yielded a cell suspension containing spindle, Y and stellate-shaped cells. There were no round cells in this suspension (Fig. 3b). The number of spindle and stellate-shaped cells in aortas of 18- to 28-week-old embryos was significantly greater compared with aortas of 5- to 12-week-old embryos. The medial layer adjacent to the IEL predominantly contained cells with processes and smooth surface covered with occasional cavellas. The processes of the majority of these cells contacted with each other. However, blind cellular extensions were essentially frequent (Fig. 3c). In the deeper (intermediate and outer) layers of media,
cell packing, shape and microrelief differed considerably from those described previously. Almost all cells were spindle shaped. There were long longitudinal creases and rows of round pits on the cell surface. Cells were densely packed and contacted by their side surfaces, creases or cell surfaces overlaying each other in a tilelike manner. Interestingly, several neighboring cells formed complexes (bundles) within which they were arranged in parallel rows. The complexes were oriented at an angle to each other (Fig. 3d).
Imunocytochemical analysis of 18-28-weeks-old embryo aortas Intimal and medial cells were intensely stained with antiactin (Fig. 3e) and anti-myosin (not illustrated) antibodies in the aortas 18- to 28-week-old embryos. Occasionally cells in the adventitia and adjacent lymph nodes were reactive with anti-macrophage antibodies. Morphologically, they were typical monocytes (not illustrated). Cells of embryonic aorta contained no macrophage antigens.
Discussion In this study, we have demonstrated that embryonic human aorta contains SMC differing in their ultrastructure, shape, packing and surface microrelief. Previously we reported on the heterogeneity on the intimal cell population in adult human aorta (Orekhov et al., 1986). Both embryonic and adult aortas contain elongated and stellate SMC. This study shows that the morphological basement of this phenomenon is laid at the 18- to 28-week-old of embryogenesis. Cells located in different sublayers of embryonic aortic media have varied shape and form variety of contacts with each other. Interestingly, SMC of the media closest to lumen have free (noncontacting) processes which can penetrate into the intima. This may be either associated with the aortic intima formation, or may reflect the processes of cell migration from the intima to the media. It is known that in human aorta, intimal cells appear in the early prenatal period (Fruntash, 1982; Kamenskaya, 1959). Their nature and origin have not been investigated. Intimal cells are directly connected with medial SMC via the internal elastic membrane. It may be suggested that the cells appear in the intima as a result of cell migration from the media. In embryonic intima we found separated or clustered cells. We assumed that one cell may migrate to the intima from the media, start dividing and form a cluster. As we have shown in present work all subendothelial cells of embryonic aorta contain SMC antigens.
Fig. 3 Structure of media in 18-28-week-old embryonic aorta. (a) Ultrastructure of SMC. t - microfilament bundle. TEM, x 23 000. (b) Cell suspension. Alcohol-alkaline dissociation, x 400. (c) SMC with blind processes, (t~). SEM, x 2000. (d) SMC forming strata in the inner layer of the media (*), oriented at an angle. SEM, x 400. (e) Immunocytochemical identification of medial aortic cells, smooth muscle ~-actin, for detection ABC-method (avidin-biotin-complex) was used, x 200.
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Irrespective of this fact, predominantly two cell populations consisting of spherical and elongated cells were isolated from human aortas at early stages of embryogenesis. Spherical and elongated cells differed ultrastructurally; the former were poorly differentiated and the latter had the major characteristics of differentiated SMC. The two cell populations differed in the character of cell packing. Spherical cells were densely packed, the connective tissue between them was practically absent, while elongated cells were separated from each other by defined layers of connective tissue. Two cell types were also found in the aortas of 18to 28-week-old embryos. Occasionally, poorly differentiated stellate cells were located in close proximity to the endothelium. The major bulk of medial cell population consisted of spindle-shaped SMC. Intimal-like
cells with processes were suited only in the innermost part of media near the IEL. Similar location of spheroid cells of early embryo, cells with processes of relatively late embryo and subendothelial stellate cells in adult human aorta offers us to propose, that spheroid cells of early embryo m a y be precursor cells for cells with processes and stellate cells, which appear on relatively late stages of embryogenesis. ACKNOWLEDGEMENTS We thank Mr Andrey Fesenko, Dr Igor Sobenin (Institute of Experimental Cardiology, National Cardiology Research Center, Moscow, Russia) and M r Suk Yoon (University of Michigan, Ann Arbor, MI, USA) for their help in the preparation of this manuscript.
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