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Frozen-fractured images of blood capillaries in heart tissue
© 1971 by Academic Press, Inc. J. ULTRASTRUCTURE RESEARCH
35, 127-146 (1971)
127
Frozen-Fractured Images of Blood Capillaries in Heart Tissue ~ LEE V, LEAK
Department of Anatomy, Harvard Medical School and Surgical Research Laboratory of Biological Structure, Massachusetts General Hospital and Shriners Burns Institute Boston Unit Received July 22, 1970 The ultrastructure of the mammalian heart capillary has been studied with the technique of freeze-etching which lends itself as a very useful method for studies designed to investigate the specialized surface features of the endothelial plasma membrane. Plasmalemmal invaginations have been shown to populate the endothelial membrane by many investigators using conventional procedures of electron microscopy (chemical fixation and plastic embedding). The method of freezeetching, however, allows a more critical evaluation of the organization and frequency of micropinocytotic vesicles over both connective tissue and luminal surfaces of the endothelium especially in replicas which present large areas of the endothelial plasma membrane in en face views. The frequency of vesicles over the endothelial surface varies considerably from one square micron of the surface to another. However, when large areas of the endothelium are taken into consideration the frequency and distribution of vesicles for both the connective tissue front and the luminal surface of the endothelium are very similar. This observation is discussed in light of current morphological and tracer studies using conventional preparatory procedures for electron microscopy. The three-dimensional relationship of the various tunics comprising the blood capillary and its relationship to the adjoining connective tissue and muscle fibers in heart tissue is also considered. Electron microscopic examinations of blood capillaries in various regions t h r o u g h o u t the b o d y have led to the recognition of several specific types of capillaries (1, 3). It was also recognized that vessels in various regions and organs of the m a m m a l i a n b o d y possessed specific fine structural features that could be related to regional differences in the permeability of blood capillaries (15). The blood capillaries to be discussed in the present c o m m u n i c a t i o n represent those of the muscular type (1, 3, 1 Supported by a grant-in-aid from the American Heart Association, the U.S.P.H.S Grant No. A107348 and the Shriners Burns Institute Boston Unit.
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i4). Previous electron m i c r o s c o p i c studies have been carried out o n chemically fixed specimens a n d i n f o r m a t i o n on the t h r e e - d i m e n s i o n a l architecture of the cellular c o m p o n e n t s of e n d o t h e l i a l cells has been o b t a i n e d by reconstructing images f r o m serially sectioned specimens (2). I n the present study, the b l o o d capillaries of the m o u s e h e a r t were e x a m i n e d b y electron m i c r o s c o p y using the recent technique of freezeetching. A l t h o u g h this m e t h o d does n o t p e r m i t a direct visualization of the tissues to be examined, it does p r o v i d e images in which a three d i m e n s i o n a l a r r a n g e m e n t of cells a n d surface c o m p o n e n t s can be visualized at the u l t r a s t r u c t u r a l level. T h e t o p o g r a p h i c a l relationship of the v a r i o u s tunics which c o m p r i s e b l o o d capillaries a n d their surface specializations are studied in the m a m m a l i a n heart. I n a d d i t i o n , the frequency a n d d i s t r i b u t i o n of vesicles within the endothelial p l a s m a l e m m a of muscle capillaries are e x a m i n e d in t h r e e - d i m e n s i o n a l relief images.
MATERIALS AND METHODS The tissues examined in this study were for the most part obtained from hearts of young adult LAF1 mice; however, observations were also carried out on heart tissue obtained from young adult guinea pigs and rats. The observations presented in this communication are for the most part based on studies made from replicas of capillaries in heart tissues of mice. Methods. Under deep ether anesthesia the heart was removed from the pericardium after a mediasternal incision; it was then quickly severed from the major vessels and placed in Ringer solution containing 10 % glycerol. Small strips of both the right and left ventricles were cut with a sharp razor blade and placed in 20 % and 30 % glycerol in Ringer solution (all solutions are chilled in an ice bath) for 20 minutes each. Some tissues were placed in a 2 % glutaraldehyde in a 0.1 M cacodylate buffer in which 30 % glycerol was added, and the treatment with this cryoprotective agent was continued for at least 30 minutes. After treatment with either of the above procedures, the tissue was placed in a copper specimen holder and quickly frozen in liquid Freon 12 that was supercooled with liquid nitrogen (9). The frozen tissue was placed in a McAlear Kreutziger freeze-etch device that had been modified by the installation of a thermocouple and heating device as described previously (9). The tissue was fractured at - 100°C and allowed to etch for varying periods (30 seconds up to about 2 minutes) and replicas were made by depositing a carbon platinum film over the frozen-fractured tissue surface (10). After the removal of tissue (8) the replicas were Large arrows indicate the direction of shadow. Fro. 1. Electron micrograph of a carbon platinum replica from a frozen fractured preparation of mouse heart. The tissue has been cleaved in such a way that the fracture plane follows the extracellular surface of the endothelium (ex-E) along its longitudinal aspects for some distance before fracturing across the interior of the capillary, revealing parts of its cytoplasm (cy), its lumen (L), and a small portion of a red blood cell (RBC). The surface of the endothelium which faces the connective tissue is covered with numerous invaginations (v). Areas of overlay between opposing terminal cell margins are seen in surface view (j). There is a narrow border along the terminal cell margin which lacks plasmalemmal invaginations (*) part of a pericyte (L) lies alongside the capillary wall and is separated from it by a basement lamina (bl). x 15 500.
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washed and mounted on uncoated 300- or 400-mesh grids? Occasionally, 150-mesh grids coated with a Parlodion film were used to pick up very large replicas that had withstood the tissue digestion and washing procedures without breaking into smaller pieces. Replicas were examined in a Philips E.M. 300 electron microscope.
OBSERVATIONS
Muscular type capillary Capillaries in heart tissue are representative of the muscular type and contain three layers within their walls, i.e., inner, middle, and outer tunics. The inner and middle tunics are continuous, while the outer tunic is very irregular as already described by a number of investigators (2, 3, 14). The inner tunic is represented by a layer of endothelial cells, and the middle layer consists of a continuous basal lamina (basement membrane) which also encompasses pericytes that may in some cases be regularly arranged along the length of the capillary wall (2). The tunica adventitia is made up of an occasional fibroblast, macrophages, and connective tissue fibers that are closely applied to the capillary wall. In an attempt to study only the vessels of the capillary bed, the observations in this investigation were generally restricted to vessels with a diameter of 7.5 to 8.5 #.
The inner tunic In the muscle type blood capillary, the inner tunic consists of a single layer of flattened endothelial cells which are closely applied to each other at their terminal margins to form a continuous layer whose inner surface (i.e., luminal surface) is in contact with the circulating blood. Each endothelial cell is bounded by a plasmalemma, and frozen-etch replicas demonstrate both extracellular and cytoplasmic surfaces as well as cross views of its limiting membrane (Figs. 1, 4, 5, and 14). The endothelial wall measures up to approximately 0.4 # in areas which contain a nucleus (Fig. 8) and may be less than 0.1 # in other regions (Fig. 13).
Surface views of the endothelial plasmalemma The nature of the fracture process allows frozen tissue to cleave or break along planes of least resistance, therefore the fracture plane may often follow the extra1 Picking up washed replicas on uncoated copper grids is facilitated if the surface tension is reduced. This is accomplished by first dipping the grid into 50 % nitric acid and rinsing very quickly by plunging it into distilled H20 several times before attempting to position the replicas on the grid. FIos. 2 and 3. The topographical relationship between opposing endothelial cell margins (j) is illustrated in surface views in these electron micrographs. In addition the distribution of plasmalemmal invagination (v) over the surface of the endothelial cell which faces the connective tissue front is also demonstrated in en face views. Fig. 2, x 13 500; Fig. 3, × 12 000.
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TABLE I FREQUENCY OF PLASMALEMMAL INVAGINATIONS OVER ENDOTHELIAL SURFACE
Endothelial Surface
Extracellular surface of connective tissue front Extracellular surface of perinuclear area Cytoplasmic surface of connective tissue front endothelium Luminal surface
Total Vesicles Counted
Total Surface Area (bt)
Average No. of Vesicles/# 2
6 965
6 343
37
178
25
36
2 021 144
1 313
40 39
25
cellular surface for a long distance before a cross fracture is made t h r o u g h the interior of the cell. T h u s m a n y of the replicas of heart muscle capillaries c o n t a i n large areas which depict en face views of the endothelial p l a s m a l e m m a (Figs. 1 a n d 3).
Distribution of vesicles over endothelial surface The precise a r r a n g e m e n t a n d d i s t r i b u t i o n of micropinocytotic vesicles over the endothelial plasma m e m b r a n e are visualized o n b o t h the extracellular a n d cytoplasmic surfaces. These micropinocytotic vesicles are a characteristic feature of the capillary endothelial cell (3, 13) a n d p o p u l a t e b o t h the l u m i n a l a n d a b l u m i n a l aspects of the endothelial cells in addition to lying within the cytoplasm (Figs. 1, 5, a n d 6). C o u n t s of vesicles over the a b l u m i n a l surface vary f r o m 20 to 70 per square m i c r o n with a n average frequency of 37 vesicles per square micron. I n v a g i n a t i o n s of the endothelial p l a s m a l e m m a are also observed in cross fracture a n d are present in b o t h t h i n a n d thick segments of the e n d o t h e l i u m (see Table I). I n addition, the threeFIGS. 4 and 5. The tubular nature of the heart muscle blood capillary is shown in three-dimensional relief in the micrographs depicted here. In each case a red blood cell (RBC) was fractured, and in Fig. 4 its position within the capillary lumen (L) surrounded by plasma (*) is readily appreciated. The frequency of plasmalemmal vesicles (v) over the extracellular (ex-pl) cytoplasmic (c-pl) and luminal (l-pl) surfaces of the endothelium is demonstrated. Fig. 4, × 22 500; Fig. 5, x l l 000. FIG. 6. The blood capillary depicted in this micrograph shows a vessel which was cross fractured over three-quarters of its circumference leaving a long segment of the abluminal plasmalemma that was fractured along its cytoplasmic surface (c-pl) over which numerous plasmalemmal invaginations (v) appear as elevations since they project toward the cytoplasm. A fractured red blood cell (RBC) occupies most of the capillary lumen. Vesicles and other cytoplasmic organelles (*) are observed in the cross fractured endothelial cytoplasm. Several collagen bundles (C) in the adventitia have been cross fractured and project above the fracture plane. Myofibers (mr) are recognized surrounding the blood capillary, x 18 000. FIG. 7. In this micrograph the replica depicts a blood capillary that is partially collapsed. Note the distance between the myofibers (rnf) and the capillary wall in the lower parts of the micrograph (arrows). Plasmalemmal invaginations (v) are illustrated in en face views along the extracellular (ex-pl) and luminal (l-pl) surfaces. In addition various profiles of vesicles (*) are observed in the endothelial cytoplasm. A small part of a cell presumed to be a pericyte (P) appears in the lower left side of the micrograph, x 11 000.
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dimensional arrangement of these invaginating vesicles which project into the cytoplasm is illustrated in replicas in which the fracture plane has coursed between the abluminal endothelial plasmalemma and the cytoplasm as demonstrated in Fig. 6. Counts made of vesicles over the cytoplasmic surface of the endothelial plasmalemma show that the average number of plasmalemmal invaginations is 40//z z. The difference in counts between the two surfaces (i.e., extracellular and cytoplasmic) could be attributed to the fact that m a n y of the connective tissue surfaces are perhaps plugged or covered by basal membrane materials; in addition, replicas of projectioning structures are more pronounced than cavities or invaginations, therefore counts of projections for the cytoplasmic surfaces are easier to determine. The relationship of the vesicles to the plasmalemma is also demonstrated in cross fracture, i.e., invaginations are often continuous with the plasmalemma. Likewise, many of the vesicles in the cytoplasm appear to be completely separated f r o m the plasmalemma. There are also vesicles that fall within the complete range of intermediates between a slightly invaginated membrane to individual vesicles that are apparently free within the cytoplasmic matrix of the endothelium (Figs. 6, 7, 9, and 13). The frequency of vesicles over the thicker regions of the endothelium, i.e., the perinuclear area, also approaches that observed over the much thinner segments (Table I). The plasmalemmal invaginations contain a substance very similar to that which occupies the subendothelial space separating the basement lamina f r o m the endothelium (Figs. 3 and 7). Numerous vesicles also populate the luminal aspect of the endothelial plasmalemma (Figs. 5 and 7). F r o m counts in the present study the frequency of vesicles over the endothelial blood front averages 39/# 2. The topographical relationship between plasmalemmal invaginations and vesicles deep within the cytoplasm is shown to good advantage in cross fractures of the capillary in which both endothelial (cytoplasmic and extracellular) surfaces are visualized in the same replica (Figs. 4 and 5). Cytoplasm
The major organelles and components recognized in the cytoplasm include the nucleus, mitochondria, membranous profiles, and large vesicles. The nucleus, which FIG. 8. The surface of the endothelium over the perinuclear region is demonstrated in this electron micrograph. The distribution of plasmalemmal vesicles (v) over the perikaryon can be appreciated in this replica. The nucleus (n) of the endothelium is also shown to good advantage. Most of the lumen (L) is occluded by a red blood cell (RBC) which has been cross fractured, x 20 000. FIG. 9. The tissue from which the replica in this micrograph was obtained was fixed in glutaraldehyde prior to freeze etching. The basement lamina (bl) of the blood capillary as well as the myofiber can be seen in this electron micrograph. The lumen (L) of this vessel is patent and a close association with the surrounding myofibers (mr) is also maintained. Profiles of vesicles (v) and membranes presumed to represent endoplasmic reticulum (er) appear in the endothelial cytoplasm, x 17 500.
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bulges into the capillary lumen, is often fractured along the perinuclear cisterna (Fig. 8). Mitochondria are often fractured along their surfaces and appear as elongated or rodlike structures; the cleavage plane may pass through the interior of these organelles revealing the arrangement of the inner membrane as it forms cisternae within their inner compartments. Profiles of membranes which are presumed to represent images of the endoplasmic reticulum and elements of the Golgi complex are also encountered in replicas of heart capillaries (Figs. 6 and 9). Structures slightly larger than the micropinocytotic vesicles were also observed and are reminiscent of a fusion product of several smaller vesicles observed in thin sections of blood capillaries. Cytoplasmic projections often extend into the lumen (Figs. 12 and 14). These are of variable lengths and seem in the majority of cases to be extensions of the terminal margins as noted earlier in thin sections of capillaries by Fawcett (3); although the abluminal surface lacks extensive marginal folds, short blebs are occasionally encountered along the tissue front of the muscle capillary.
Endothelial intercellular junctions Terminal margins of adjacent endothelial cells overlap each other or they are held in close apposition by an imbrication of the endothelial cell processes (Figs. 1, 2, and 10-12). Cell junctions of adjacent endothelial cells are also observed in surface views as demonstrated in Figs. 1-3. An area of about 0.4 # wide is seen along the terminal cell margins which is free of plasmalemmal invaginations (Fig. 1). Cross fracture of cell junctions indicate that the area of overlap between opposing cells is quite variable (cf. Figs. 1 and 2 with 10-12).
The middle layer of tunic The components of this layer include a basement lamina (basement membrane) and occasional pericytes which have been recently shown to be included within the basement lamina (2, 3).
Basement lamina This thin layer is not always recognized in replicas of frozen-etched specimens. However, with deep etching, a thin line consisting of small irregular filaments and granules forms a continuous band around the capillary wall. In specimens fixed prior to freeze-etching, the boundaries of a basement lamina are more pronounced, FIGS. 10-12. The relationship between opposing endothelial cell margins is demonstrated in these electron micrographs. In Fig. 10, the adjacent endothelial cells are held in close opposition by a simple abutment. In Fig. 11 there is an imbrication of the terminal cell margins while in Fig. 12 there is an overlapping of the opposing cell margins. Figs. 10-12, x 48 000.
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and a well defined layer of a filamentous or granular nature is separated from the endothelial surface by a narrow space which appears to be free of formed elements (Fig. 9). A narrow subendothelial space is also recognized in thin sections obtained by conventional methods for electron microscopy. The appearance of this space has been attributed to shrinkage during the preparation procedures (2). The intimate association of thin filaments and collagen fibrils at the periphery of the basement lamina is recognized when these extracellular components are cross fractured. These very often project above the fracture plane (Fig. 6).
Pericytes These cells are intimately associated with the abluminal aspect of the capillary wall and have been demonstrated by conventional methods of electron microscopy to be included within the basement lamina (Figs. 1, 7, 15, 16).
The outer tunic or adventitia The outermost layer of the capillary wall in heart muscles is discontinuous and consists of connective tissue cells and extracellular elements. It has been determined from thin sections that fibroblasts and macrophages comprise the major cell types associated with this outer layer (14). Both cellular and extracellular elements lie outside of the basement lamina (Figs. 6, 7, and 16).
DISCUSSION In this investigation the observations demonstrate that the technique of freezeetching lends itself as a very useful method for identifying specialized surface features possessed by the various tunics of the mammalian blood capillary. The observations described above depict the three-dimensional organization of the muscular type blood capillary and the specific surface features possessed by the concentric layers which comprise the walls of these vessels.
FIG. 13. In this micrograph the capillary lumen (L) is distended and contains a red blood cell (RBC). The cytoplasmic surface of the abluminal endothelial plasmalemma reveals invaginations at various points (arrows). A cell junction (j) is seen on the lower left side of this micrograph. Vesicles in lumen are presumed to represent platelets (*). x 42 500. FIG. 14. This micrograph depicts a replica of a capillary from an unfixed preparation of heart tissue. The close relation between the capillary wall and surrounding myofibers (mf) has been preserved (arrows). Several endothelial marginal folds (ef) extend into the lumen (L). x 40 000. FIG. 15. The intimate relationship between the capillary and pericyte (P) is demonstrated in this micrograph. The fracture plane has passed along the external surface of the pericyte (ex-pl) for a short distance before fracturing across the cell revealing portions of its cytoplasm and nucleus (n). Various profiles of the Golgi (G) and other cytoplasmic organelles are also observed. The fracture plane has followed the capillary surface and also cross fractured the vessel, x 11 500.
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In the present study large surface areas of the endothelial cell could be obtained for a critical evaluation of the precise distribution and organization of plasmalemmal invaginations which populate both luminal and abluminal surfaces of the endothelial cell. Our observations show that the number of plasmalemmal invaginations over the endothelial surface is quite varied, ranging from 20 to 70 vesicles per square micron of the surface area. It should be emphasized that these counts are only of plasmalemmal invaginations and do not represent intact vesicles within the cytoplasm of the endothelial cells. It is of special interest to note, however, that the concentration of invaginations was very similar for both the attenuated regions as well as the thick (i.e., perinuclear) areas of the endothelium. In addition, a similar distribution of vesicles was also noted for the cytoplasmic surfaces of the endothelial plasmalemma which provides additional support for the counts obtained for extracellular or connective tissue surfaces of the endothelial cells. It is of further interest to note that the counts of plasmalemmal invaginations over the luminal surfaces of the endothelial cells indicate that the distribution of these vesicles are comparable to those of the connective tissue front. Although there is variability in the number of plasmalemmal invaginations from one area of the endothelium to another, the average frequency for large surface areas of both the attenuated regions as well as the perinuclear surface areas of the endothelium are relatively constant, that is, in the neighborhood of 38 vesicles per square micron. Since plasmalemmal vesicles were first implicated as a means of transport in blood capillaries (11) a large number of morphological and tracer studies have subsequently demonstrated the role of these vesicles in the transport of large molecules and colloidal particles up to 300 A (i.e., tracer particles) from the luminal to the connective tissue front (2, 4-7, 12-14). Counts of endothelial vesicles made by Bruns and Palade (2) suggested that the frequency of plasmalemmal vesicles is slightly higher for the attenuated areas of the connective tissue front than for the perinuclear regions of the endothelial cell. The data obtained in the present study indicate that when large surface areas are used for evaluating the distribution of plasmalemmal invaginations over the endothelial surfaces the average frequencies for both surfaces are similar. Although a large number of replicas were studied from unfixed and fixed preparations, none contained a single plasmalemmal vesicle which bridged both connective FIG. 16. The three dimensional architecture of the heart muscle capillary with its luminal content of red blood cells (RBC) and wall of endothelium (E), basement lamina (bl) and pericyte (P) is illustrated in this diagram. This diagram was constructed from three dimensional relief images obtained from replicas of heart capillaries of both fixed and unfixed frozen etched preparations. Both luminal and connective tissue surfaces of the endothelium are populated by plasmalemmal invaginations (micropinocytotic vesicles). Cut away segments of the endothelium depict the appearance of these vesicles (v) over the cytoplasmic surface. The topographical association between opposing cell margins to form intercellular junctions (]) is depicted in both surface and cross views. The basement lamina (bl) is continuous over the endothelial surface as well as the adjacent pericyte (P).
FROZEN-FRACTURED IMAGES OF BLOOD CAPILLARIES IN HEART TISSUE
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tissue and luminal surfaces of the endothelial cell to form an uninterrupted channel. Three-dimensional reconstructions from serial sections of muscular blood capillaries (2) over approximately 1.33 microns long, approximately 0.33 micron wide and approximately 0.30 micron thick, also failed to demonstrate continuous plasmalemmal vesicles across the endothelium. Since replicas of frozen-etched preparations are indicative of the natural state of tissues the striking similarity between the images obtained in the present study and those observed from conventional preparations (i.e., chemical fixation and plastic embedding) corroborates the distribution of plasmalemmal invagination and the distribution of vesicles throughout the cytoplasm of the endothelial cell of heart muscle capillaries. The three-dimensional relationship of the various tunics comprising the muscular capillary wall can be summarized in the diagram depicted in Fig. 16. The organization and distribution of plasmalemmal invaginations along both luminal and connective tissue fronts of the endothelial surfaces as well as the cytoplasmic surfaces of the two limiting membranes are illustrated. Replicas obtained from frozen etched preparations in the present study as well as the images seen in conventional preparations for electron microscopy (2, 6, 7) demonstrate that plasmalemmal vesicles always invaginate toward the cytoplasm whether from the connective tissue or the luminal front. REFERENCES 1. BENNETT,H. S., LUFT, J. H. and HAMPTON,J. C., Amer. J. Physiol. 196, 381 (1959). 2. BRUNS, R. R. and PALADE,G. E., J. Cell Biol. 37, 244 (1968). 3. FAWCETT,D. W., in ORBISON, J. L. and SMITH, D. E. (Eds.), The Peripheral Blood Vessels. Williams and Wilkins, Baltimore, Maryland, 1963. 4. JENNINGS, M. A., MARCHESI, V. T. and FLOREY, H., Proc. Roy. Soc. Ser. B 156, 14 (1962). 5. JENNINGS, M. A. and FLOREY, H., Proc. Roy. Soc. Ser. B 167, 39 (1967). 6. KARNOVSKY,M. J., J. Cell Biol. 27, 49A (1965). 7. - ibid. 35, 213 (1967). 8. LEAK, L. V., J. Ultrastruct. Res. 25, 253 (1968). 9. - ibid. 31, 76 (1970). 10. MOOR, H. and MOnLETHALER,K., J. Cell Biol. 17, 609 (1963). 11. PALADE,G. E., J. Appl. Phys. 24, 1424 (1953). 12. - Anat. Rec. 136, 254 (1960). 13. - Circulation 24, 368 (1961). 14. PALADE, G. E. and BRUNS, R. R., in SIPERSTEIN, M. D., COLWELL,A. R., SR. and MEYER, K. (Eds.), Small Blood Vessel Involvement in Diabetes Mellitus, p. 39. American Institute of Biological Sciences, Washington, D.C., 1964. 15. PAPPENnEIMER,J. R., Physiol. Rev. 33, 387 (1953).
35, 127-146 (1971)
127
Frozen-Fractured Images of Blood Capillaries in Heart Tissue ~ LEE V, LEAK
Department of Anatomy, Harvard Medical School and Surgical Research Laboratory of Biological Structure, Massachusetts General Hospital and Shriners Burns Institute Boston Unit Received July 22, 1970 The ultrastructure of the mammalian heart capillary has been studied with the technique of freeze-etching which lends itself as a very useful method for studies designed to investigate the specialized surface features of the endothelial plasma membrane. Plasmalemmal invaginations have been shown to populate the endothelial membrane by many investigators using conventional procedures of electron microscopy (chemical fixation and plastic embedding). The method of freezeetching, however, allows a more critical evaluation of the organization and frequency of micropinocytotic vesicles over both connective tissue and luminal surfaces of the endothelium especially in replicas which present large areas of the endothelial plasma membrane in en face views. The frequency of vesicles over the endothelial surface varies considerably from one square micron of the surface to another. However, when large areas of the endothelium are taken into consideration the frequency and distribution of vesicles for both the connective tissue front and the luminal surface of the endothelium are very similar. This observation is discussed in light of current morphological and tracer studies using conventional preparatory procedures for electron microscopy. The three-dimensional relationship of the various tunics comprising the blood capillary and its relationship to the adjoining connective tissue and muscle fibers in heart tissue is also considered. Electron microscopic examinations of blood capillaries in various regions t h r o u g h o u t the b o d y have led to the recognition of several specific types of capillaries (1, 3). It was also recognized that vessels in various regions and organs of the m a m m a l i a n b o d y possessed specific fine structural features that could be related to regional differences in the permeability of blood capillaries (15). The blood capillaries to be discussed in the present c o m m u n i c a t i o n represent those of the muscular type (1, 3, 1 Supported by a grant-in-aid from the American Heart Association, the U.S.P.H.S Grant No. A107348 and the Shriners Burns Institute Boston Unit.
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i4). Previous electron m i c r o s c o p i c studies have been carried out o n chemically fixed specimens a n d i n f o r m a t i o n on the t h r e e - d i m e n s i o n a l architecture of the cellular c o m p o n e n t s of e n d o t h e l i a l cells has been o b t a i n e d by reconstructing images f r o m serially sectioned specimens (2). I n the present study, the b l o o d capillaries of the m o u s e h e a r t were e x a m i n e d b y electron m i c r o s c o p y using the recent technique of freezeetching. A l t h o u g h this m e t h o d does n o t p e r m i t a direct visualization of the tissues to be examined, it does p r o v i d e images in which a three d i m e n s i o n a l a r r a n g e m e n t of cells a n d surface c o m p o n e n t s can be visualized at the u l t r a s t r u c t u r a l level. T h e t o p o g r a p h i c a l relationship of the v a r i o u s tunics which c o m p r i s e b l o o d capillaries a n d their surface specializations are studied in the m a m m a l i a n heart. I n a d d i t i o n , the frequency a n d d i s t r i b u t i o n of vesicles within the endothelial p l a s m a l e m m a of muscle capillaries are e x a m i n e d in t h r e e - d i m e n s i o n a l relief images.
MATERIALS AND METHODS The tissues examined in this study were for the most part obtained from hearts of young adult LAF1 mice; however, observations were also carried out on heart tissue obtained from young adult guinea pigs and rats. The observations presented in this communication are for the most part based on studies made from replicas of capillaries in heart tissues of mice. Methods. Under deep ether anesthesia the heart was removed from the pericardium after a mediasternal incision; it was then quickly severed from the major vessels and placed in Ringer solution containing 10 % glycerol. Small strips of both the right and left ventricles were cut with a sharp razor blade and placed in 20 % and 30 % glycerol in Ringer solution (all solutions are chilled in an ice bath) for 20 minutes each. Some tissues were placed in a 2 % glutaraldehyde in a 0.1 M cacodylate buffer in which 30 % glycerol was added, and the treatment with this cryoprotective agent was continued for at least 30 minutes. After treatment with either of the above procedures, the tissue was placed in a copper specimen holder and quickly frozen in liquid Freon 12 that was supercooled with liquid nitrogen (9). The frozen tissue was placed in a McAlear Kreutziger freeze-etch device that had been modified by the installation of a thermocouple and heating device as described previously (9). The tissue was fractured at - 100°C and allowed to etch for varying periods (30 seconds up to about 2 minutes) and replicas were made by depositing a carbon platinum film over the frozen-fractured tissue surface (10). After the removal of tissue (8) the replicas were Large arrows indicate the direction of shadow. Fro. 1. Electron micrograph of a carbon platinum replica from a frozen fractured preparation of mouse heart. The tissue has been cleaved in such a way that the fracture plane follows the extracellular surface of the endothelium (ex-E) along its longitudinal aspects for some distance before fracturing across the interior of the capillary, revealing parts of its cytoplasm (cy), its lumen (L), and a small portion of a red blood cell (RBC). The surface of the endothelium which faces the connective tissue is covered with numerous invaginations (v). Areas of overlay between opposing terminal cell margins are seen in surface view (j). There is a narrow border along the terminal cell margin which lacks plasmalemmal invaginations (*) part of a pericyte (L) lies alongside the capillary wall and is separated from it by a basement lamina (bl). x 15 500.
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washed and mounted on uncoated 300- or 400-mesh grids? Occasionally, 150-mesh grids coated with a Parlodion film were used to pick up very large replicas that had withstood the tissue digestion and washing procedures without breaking into smaller pieces. Replicas were examined in a Philips E.M. 300 electron microscope.
OBSERVATIONS
Muscular type capillary Capillaries in heart tissue are representative of the muscular type and contain three layers within their walls, i.e., inner, middle, and outer tunics. The inner and middle tunics are continuous, while the outer tunic is very irregular as already described by a number of investigators (2, 3, 14). The inner tunic is represented by a layer of endothelial cells, and the middle layer consists of a continuous basal lamina (basement membrane) which also encompasses pericytes that may in some cases be regularly arranged along the length of the capillary wall (2). The tunica adventitia is made up of an occasional fibroblast, macrophages, and connective tissue fibers that are closely applied to the capillary wall. In an attempt to study only the vessels of the capillary bed, the observations in this investigation were generally restricted to vessels with a diameter of 7.5 to 8.5 #.
The inner tunic In the muscle type blood capillary, the inner tunic consists of a single layer of flattened endothelial cells which are closely applied to each other at their terminal margins to form a continuous layer whose inner surface (i.e., luminal surface) is in contact with the circulating blood. Each endothelial cell is bounded by a plasmalemma, and frozen-etch replicas demonstrate both extracellular and cytoplasmic surfaces as well as cross views of its limiting membrane (Figs. 1, 4, 5, and 14). The endothelial wall measures up to approximately 0.4 # in areas which contain a nucleus (Fig. 8) and may be less than 0.1 # in other regions (Fig. 13).
Surface views of the endothelial plasmalemma The nature of the fracture process allows frozen tissue to cleave or break along planes of least resistance, therefore the fracture plane may often follow the extra1 Picking up washed replicas on uncoated copper grids is facilitated if the surface tension is reduced. This is accomplished by first dipping the grid into 50 % nitric acid and rinsing very quickly by plunging it into distilled H20 several times before attempting to position the replicas on the grid. FIos. 2 and 3. The topographical relationship between opposing endothelial cell margins (j) is illustrated in surface views in these electron micrographs. In addition the distribution of plasmalemmal invagination (v) over the surface of the endothelial cell which faces the connective tissue front is also demonstrated in en face views. Fig. 2, x 13 500; Fig. 3, × 12 000.
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TABLE I FREQUENCY OF PLASMALEMMAL INVAGINATIONS OVER ENDOTHELIAL SURFACE
Endothelial Surface
Extracellular surface of connective tissue front Extracellular surface of perinuclear area Cytoplasmic surface of connective tissue front endothelium Luminal surface
Total Vesicles Counted
Total Surface Area (bt)
Average No. of Vesicles/# 2
6 965
6 343
37
178
25
36
2 021 144
1 313
40 39
25
cellular surface for a long distance before a cross fracture is made t h r o u g h the interior of the cell. T h u s m a n y of the replicas of heart muscle capillaries c o n t a i n large areas which depict en face views of the endothelial p l a s m a l e m m a (Figs. 1 a n d 3).
Distribution of vesicles over endothelial surface The precise a r r a n g e m e n t a n d d i s t r i b u t i o n of micropinocytotic vesicles over the endothelial plasma m e m b r a n e are visualized o n b o t h the extracellular a n d cytoplasmic surfaces. These micropinocytotic vesicles are a characteristic feature of the capillary endothelial cell (3, 13) a n d p o p u l a t e b o t h the l u m i n a l a n d a b l u m i n a l aspects of the endothelial cells in addition to lying within the cytoplasm (Figs. 1, 5, a n d 6). C o u n t s of vesicles over the a b l u m i n a l surface vary f r o m 20 to 70 per square m i c r o n with a n average frequency of 37 vesicles per square micron. I n v a g i n a t i o n s of the endothelial p l a s m a l e m m a are also observed in cross fracture a n d are present in b o t h t h i n a n d thick segments of the e n d o t h e l i u m (see Table I). I n addition, the threeFIGS. 4 and 5. The tubular nature of the heart muscle blood capillary is shown in three-dimensional relief in the micrographs depicted here. In each case a red blood cell (RBC) was fractured, and in Fig. 4 its position within the capillary lumen (L) surrounded by plasma (*) is readily appreciated. The frequency of plasmalemmal vesicles (v) over the extracellular (ex-pl) cytoplasmic (c-pl) and luminal (l-pl) surfaces of the endothelium is demonstrated. Fig. 4, × 22 500; Fig. 5, x l l 000. FIG. 6. The blood capillary depicted in this micrograph shows a vessel which was cross fractured over three-quarters of its circumference leaving a long segment of the abluminal plasmalemma that was fractured along its cytoplasmic surface (c-pl) over which numerous plasmalemmal invaginations (v) appear as elevations since they project toward the cytoplasm. A fractured red blood cell (RBC) occupies most of the capillary lumen. Vesicles and other cytoplasmic organelles (*) are observed in the cross fractured endothelial cytoplasm. Several collagen bundles (C) in the adventitia have been cross fractured and project above the fracture plane. Myofibers (mr) are recognized surrounding the blood capillary, x 18 000. FIG. 7. In this micrograph the replica depicts a blood capillary that is partially collapsed. Note the distance between the myofibers (rnf) and the capillary wall in the lower parts of the micrograph (arrows). Plasmalemmal invaginations (v) are illustrated in en face views along the extracellular (ex-pl) and luminal (l-pl) surfaces. In addition various profiles of vesicles (*) are observed in the endothelial cytoplasm. A small part of a cell presumed to be a pericyte (P) appears in the lower left side of the micrograph, x 11 000.
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dimensional arrangement of these invaginating vesicles which project into the cytoplasm is illustrated in replicas in which the fracture plane has coursed between the abluminal endothelial plasmalemma and the cytoplasm as demonstrated in Fig. 6. Counts made of vesicles over the cytoplasmic surface of the endothelial plasmalemma show that the average number of plasmalemmal invaginations is 40//z z. The difference in counts between the two surfaces (i.e., extracellular and cytoplasmic) could be attributed to the fact that m a n y of the connective tissue surfaces are perhaps plugged or covered by basal membrane materials; in addition, replicas of projectioning structures are more pronounced than cavities or invaginations, therefore counts of projections for the cytoplasmic surfaces are easier to determine. The relationship of the vesicles to the plasmalemma is also demonstrated in cross fracture, i.e., invaginations are often continuous with the plasmalemma. Likewise, many of the vesicles in the cytoplasm appear to be completely separated f r o m the plasmalemma. There are also vesicles that fall within the complete range of intermediates between a slightly invaginated membrane to individual vesicles that are apparently free within the cytoplasmic matrix of the endothelium (Figs. 6, 7, 9, and 13). The frequency of vesicles over the thicker regions of the endothelium, i.e., the perinuclear area, also approaches that observed over the much thinner segments (Table I). The plasmalemmal invaginations contain a substance very similar to that which occupies the subendothelial space separating the basement lamina f r o m the endothelium (Figs. 3 and 7). Numerous vesicles also populate the luminal aspect of the endothelial plasmalemma (Figs. 5 and 7). F r o m counts in the present study the frequency of vesicles over the endothelial blood front averages 39/# 2. The topographical relationship between plasmalemmal invaginations and vesicles deep within the cytoplasm is shown to good advantage in cross fractures of the capillary in which both endothelial (cytoplasmic and extracellular) surfaces are visualized in the same replica (Figs. 4 and 5). Cytoplasm
The major organelles and components recognized in the cytoplasm include the nucleus, mitochondria, membranous profiles, and large vesicles. The nucleus, which FIG. 8. The surface of the endothelium over the perinuclear region is demonstrated in this electron micrograph. The distribution of plasmalemmal vesicles (v) over the perikaryon can be appreciated in this replica. The nucleus (n) of the endothelium is also shown to good advantage. Most of the lumen (L) is occluded by a red blood cell (RBC) which has been cross fractured, x 20 000. FIG. 9. The tissue from which the replica in this micrograph was obtained was fixed in glutaraldehyde prior to freeze etching. The basement lamina (bl) of the blood capillary as well as the myofiber can be seen in this electron micrograph. The lumen (L) of this vessel is patent and a close association with the surrounding myofibers (mr) is also maintained. Profiles of vesicles (v) and membranes presumed to represent endoplasmic reticulum (er) appear in the endothelial cytoplasm, x 17 500.
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bulges into the capillary lumen, is often fractured along the perinuclear cisterna (Fig. 8). Mitochondria are often fractured along their surfaces and appear as elongated or rodlike structures; the cleavage plane may pass through the interior of these organelles revealing the arrangement of the inner membrane as it forms cisternae within their inner compartments. Profiles of membranes which are presumed to represent images of the endoplasmic reticulum and elements of the Golgi complex are also encountered in replicas of heart capillaries (Figs. 6 and 9). Structures slightly larger than the micropinocytotic vesicles were also observed and are reminiscent of a fusion product of several smaller vesicles observed in thin sections of blood capillaries. Cytoplasmic projections often extend into the lumen (Figs. 12 and 14). These are of variable lengths and seem in the majority of cases to be extensions of the terminal margins as noted earlier in thin sections of capillaries by Fawcett (3); although the abluminal surface lacks extensive marginal folds, short blebs are occasionally encountered along the tissue front of the muscle capillary.
Endothelial intercellular junctions Terminal margins of adjacent endothelial cells overlap each other or they are held in close apposition by an imbrication of the endothelial cell processes (Figs. 1, 2, and 10-12). Cell junctions of adjacent endothelial cells are also observed in surface views as demonstrated in Figs. 1-3. An area of about 0.4 # wide is seen along the terminal cell margins which is free of plasmalemmal invaginations (Fig. 1). Cross fracture of cell junctions indicate that the area of overlap between opposing cells is quite variable (cf. Figs. 1 and 2 with 10-12).
The middle layer of tunic The components of this layer include a basement lamina (basement membrane) and occasional pericytes which have been recently shown to be included within the basement lamina (2, 3).
Basement lamina This thin layer is not always recognized in replicas of frozen-etched specimens. However, with deep etching, a thin line consisting of small irregular filaments and granules forms a continuous band around the capillary wall. In specimens fixed prior to freeze-etching, the boundaries of a basement lamina are more pronounced, FIGS. 10-12. The relationship between opposing endothelial cell margins is demonstrated in these electron micrographs. In Fig. 10, the adjacent endothelial cells are held in close opposition by a simple abutment. In Fig. 11 there is an imbrication of the terminal cell margins while in Fig. 12 there is an overlapping of the opposing cell margins. Figs. 10-12, x 48 000.
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and a well defined layer of a filamentous or granular nature is separated from the endothelial surface by a narrow space which appears to be free of formed elements (Fig. 9). A narrow subendothelial space is also recognized in thin sections obtained by conventional methods for electron microscopy. The appearance of this space has been attributed to shrinkage during the preparation procedures (2). The intimate association of thin filaments and collagen fibrils at the periphery of the basement lamina is recognized when these extracellular components are cross fractured. These very often project above the fracture plane (Fig. 6).
Pericytes These cells are intimately associated with the abluminal aspect of the capillary wall and have been demonstrated by conventional methods of electron microscopy to be included within the basement lamina (Figs. 1, 7, 15, 16).
The outer tunic or adventitia The outermost layer of the capillary wall in heart muscles is discontinuous and consists of connective tissue cells and extracellular elements. It has been determined from thin sections that fibroblasts and macrophages comprise the major cell types associated with this outer layer (14). Both cellular and extracellular elements lie outside of the basement lamina (Figs. 6, 7, and 16).
DISCUSSION In this investigation the observations demonstrate that the technique of freezeetching lends itself as a very useful method for identifying specialized surface features possessed by the various tunics of the mammalian blood capillary. The observations described above depict the three-dimensional organization of the muscular type blood capillary and the specific surface features possessed by the concentric layers which comprise the walls of these vessels.
FIG. 13. In this micrograph the capillary lumen (L) is distended and contains a red blood cell (RBC). The cytoplasmic surface of the abluminal endothelial plasmalemma reveals invaginations at various points (arrows). A cell junction (j) is seen on the lower left side of this micrograph. Vesicles in lumen are presumed to represent platelets (*). x 42 500. FIG. 14. This micrograph depicts a replica of a capillary from an unfixed preparation of heart tissue. The close relation between the capillary wall and surrounding myofibers (mf) has been preserved (arrows). Several endothelial marginal folds (ef) extend into the lumen (L). x 40 000. FIG. 15. The intimate relationship between the capillary and pericyte (P) is demonstrated in this micrograph. The fracture plane has passed along the external surface of the pericyte (ex-pl) for a short distance before fracturing across the cell revealing portions of its cytoplasm and nucleus (n). Various profiles of the Golgi (G) and other cytoplasmic organelles are also observed. The fracture plane has followed the capillary surface and also cross fractured the vessel, x 11 500.
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In the present study large surface areas of the endothelial cell could be obtained for a critical evaluation of the precise distribution and organization of plasmalemmal invaginations which populate both luminal and abluminal surfaces of the endothelial cell. Our observations show that the number of plasmalemmal invaginations over the endothelial surface is quite varied, ranging from 20 to 70 vesicles per square micron of the surface area. It should be emphasized that these counts are only of plasmalemmal invaginations and do not represent intact vesicles within the cytoplasm of the endothelial cells. It is of special interest to note, however, that the concentration of invaginations was very similar for both the attenuated regions as well as the thick (i.e., perinuclear) areas of the endothelium. In addition, a similar distribution of vesicles was also noted for the cytoplasmic surfaces of the endothelial plasmalemma which provides additional support for the counts obtained for extracellular or connective tissue surfaces of the endothelial cells. It is of further interest to note that the counts of plasmalemmal invaginations over the luminal surfaces of the endothelial cells indicate that the distribution of these vesicles are comparable to those of the connective tissue front. Although there is variability in the number of plasmalemmal invaginations from one area of the endothelium to another, the average frequency for large surface areas of both the attenuated regions as well as the perinuclear surface areas of the endothelium are relatively constant, that is, in the neighborhood of 38 vesicles per square micron. Since plasmalemmal vesicles were first implicated as a means of transport in blood capillaries (11) a large number of morphological and tracer studies have subsequently demonstrated the role of these vesicles in the transport of large molecules and colloidal particles up to 300 A (i.e., tracer particles) from the luminal to the connective tissue front (2, 4-7, 12-14). Counts of endothelial vesicles made by Bruns and Palade (2) suggested that the frequency of plasmalemmal vesicles is slightly higher for the attenuated areas of the connective tissue front than for the perinuclear regions of the endothelial cell. The data obtained in the present study indicate that when large surface areas are used for evaluating the distribution of plasmalemmal invaginations over the endothelial surfaces the average frequencies for both surfaces are similar. Although a large number of replicas were studied from unfixed and fixed preparations, none contained a single plasmalemmal vesicle which bridged both connective FIG. 16. The three dimensional architecture of the heart muscle capillary with its luminal content of red blood cells (RBC) and wall of endothelium (E), basement lamina (bl) and pericyte (P) is illustrated in this diagram. This diagram was constructed from three dimensional relief images obtained from replicas of heart capillaries of both fixed and unfixed frozen etched preparations. Both luminal and connective tissue surfaces of the endothelium are populated by plasmalemmal invaginations (micropinocytotic vesicles). Cut away segments of the endothelium depict the appearance of these vesicles (v) over the cytoplasmic surface. The topographical association between opposing cell margins to form intercellular junctions (]) is depicted in both surface and cross views. The basement lamina (bl) is continuous over the endothelial surface as well as the adjacent pericyte (P).
FROZEN-FRACTURED IMAGES OF BLOOD CAPILLARIES IN HEART TISSUE
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tissue and luminal surfaces of the endothelial cell to form an uninterrupted channel. Three-dimensional reconstructions from serial sections of muscular blood capillaries (2) over approximately 1.33 microns long, approximately 0.33 micron wide and approximately 0.30 micron thick, also failed to demonstrate continuous plasmalemmal vesicles across the endothelium. Since replicas of frozen-etched preparations are indicative of the natural state of tissues the striking similarity between the images obtained in the present study and those observed from conventional preparations (i.e., chemical fixation and plastic embedding) corroborates the distribution of plasmalemmal invagination and the distribution of vesicles throughout the cytoplasm of the endothelial cell of heart muscle capillaries. The three-dimensional relationship of the various tunics comprising the muscular capillary wall can be summarized in the diagram depicted in Fig. 16. The organization and distribution of plasmalemmal invaginations along both luminal and connective tissue fronts of the endothelial surfaces as well as the cytoplasmic surfaces of the two limiting membranes are illustrated. Replicas obtained from frozen etched preparations in the present study as well as the images seen in conventional preparations for electron microscopy (2, 6, 7) demonstrate that plasmalemmal vesicles always invaginate toward the cytoplasm whether from the connective tissue or the luminal front. REFERENCES 1. BENNETT,H. S., LUFT, J. H. and HAMPTON,J. C., Amer. J. Physiol. 196, 381 (1959). 2. BRUNS, R. R. and PALADE,G. E., J. Cell Biol. 37, 244 (1968). 3. FAWCETT,D. W., in ORBISON, J. L. and SMITH, D. E. (Eds.), The Peripheral Blood Vessels. Williams and Wilkins, Baltimore, Maryland, 1963. 4. JENNINGS, M. A., MARCHESI, V. T. and FLOREY, H., Proc. Roy. Soc. Ser. B 156, 14 (1962). 5. JENNINGS, M. A. and FLOREY, H., Proc. Roy. Soc. Ser. B 167, 39 (1967). 6. KARNOVSKY,M. J., J. Cell Biol. 27, 49A (1965). 7. - ibid. 35, 213 (1967). 8. LEAK, L. V., J. Ultrastruct. Res. 25, 253 (1968). 9. - ibid. 31, 76 (1970). 10. MOOR, H. and MOnLETHALER,K., J. Cell Biol. 17, 609 (1963). 11. PALADE,G. E., J. Appl. Phys. 24, 1424 (1953). 12. - Anat. Rec. 136, 254 (1960). 13. - Circulation 24, 368 (1961). 14. PALADE, G. E. and BRUNS, R. R., in SIPERSTEIN, M. D., COLWELL,A. R., SR. and MEYER, K. (Eds.), Small Blood Vessel Involvement in Diabetes Mellitus, p. 39. American Institute of Biological Sciences, Washington, D.C., 1964. 15. PAPPENnEIMER,J. R., Physiol. Rev. 33, 387 (1953).