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Size and distribution of endothelial plasmalemmal vesicles in consecutive segments of the microvasculature in cat skeletal muscle
MICROVASCULAR
RESEARCH
17, 107-117
(1979)
Size and Distribution of Endothelial Plasmalemmal Vesicles in Consecutive Segments of the Microvasculature in Cat Skeletal Muscle BENCT R. JOHANSSON Department
of Anatomy,
University of Gtiteborg,
Giiteborg,
Sweden
Received September II, 1977 Vessels belonging to five individual microvascular units (arteriole-capillary-venule sequence) in the tenuissimus muscle of cat were identified in the electron microscope by utilizing a serial sectioning technique. Morphometric analyses were performed on the plasmalemmal vesicle population in four defined vessel segments: terminal arterioles, arteriolar fourth of capillaries, venular fourth of capillaries, and postcapillary venules. The sizes and numbers of vesicles, classified as luminal, abluminal, and free vesicles, were assessed. The abluminal vesicles were regularly more numerous than the luminal ones in the same segment. There were markedly different patterns of vesiculation along the five microvascular units. This finding was interpreted tentatively as indicating a reactivity of the endothelial transport function to factors of an unknown nature in the local microenvironment.
INTRODUCTION Ultrastructural studies on the microvasculature are generally performed by collecting data from not thoroughly characterized parts of the nutritive vessels. The concept “capillary” becomes a common designation for small vessels with a maximal diameter, and their location in the terminal arteriole-true capillarypostcapillary venule sequence is often uncertain. However, longitudinal, functional,. and structural differentiations do exist along the consecutive segments of the nutritive vessel. These differentiations have been detailed in intestinal villi (Casley-Smith, 1971) and in tissues with a limited extension in the third dimension both with conventional electron microscopy (Rhodin, 1967, 1968) and with freeze-fracturing techniques (Simionescu et ol., 1975). Recently, Simionescu et al. (1976) also reported variations in the structural substrate of vascular permeability in sequential segments of the microvasculature in mouse diaphragm. Their findings indicated, for example, that the rate of transendothelial transport via plasmalemmal vesicles increased toward the venular end of the capillaries (Simionescu et al., 1976). In two preceding investigations on rat skeletal muscle, I was able to demonstrate that the number, size, and distribution of endothelial plasmalemmal vesicles were different at different capillary hydrostatic pressures (Johansson, 1976,1979b). These observations thus indicated the possibility of a coupling between vesicle morphology and intravascular pressure level. The aim of the present study was to elucidate with the electron microscope whether ultrastructural differentiations exist in the endothelium, notably the plasmalemmal vesicle system, along the different microvascular segments in 107
‘X26-2862i79~020107-I 1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
108
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skeletal muscle. It was decided not to collect randomly selected vessels of defined sizes but to compare the endothelial ultrastructure of vascular segments belonging to the same microvascular unit derived from a common arteriole and ending up in a common venule. Thus, also the extremities of the capillaries were defined and compared. MATERIALS
AND
METHODS
Animals and Specimen Excision The study was performed on the tenuissimus muscle in cats. This muscle has been utilized in intravital microscopic studies of the microcirculation (Eriksson and Myrhage, 1972; Fronek and Zweifach, 1975). Five cats, body weight 2-2.5 kg, were used. The animals were anesthetized with chloralose, 50 mg/kg body weight, and were breathing spontaneously. The left tenuissimus muscle was exposed surgically according to Brinemark and Eriksson (1972). After exposing the muscle, its midportion was grasped with a clamp and simultaneously flooded with fixative. To one shank of the clamp, a 1.5 x 2-cm glass plate was adapted; to the other, a slightly smaller metallic frame. The use of a clamp allowed the muscle to be kept flat and undistorted throughout prefixation, which facilitated the subsequent identification of vascular segments. Moreover, the retained red blood cells in the vessels served as “markers” in the different steps of vessel identification. After in situ fixation for 5 min, the muscle was cut outside the frame of the clamp and the whole assembly was immersed in cold fixative for at least 2 hr. Tissue Processing and Electron Microscopy The fixative was a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.09 M Na cacodylate buffer, pH 7.2. After rinsing in fresh buffer, the midportions of the muscles were cut transversely into five to seven approximately 1.5-mm-long pieces which were postfixed in 1% 0~0, in 0.1 M Na cacodylate buffer, pH 7.2, for 2 hr. Dehydration in graded series of ethanol and embedding in Epon in flat molds were performed according to routine procedures. For all sectioning, an LKB Ultrotome with glass knives was used. Ultrathin sections were mounted on copper grids and contrasted with lead citrate and uranyl acetate. Only sections with a silver-gray interference color were collected. The sections were examined in a Philips EM 300 electron microscope. Its magnification was calibrated with a carbon grating replica, 2160 lines/mm. Identification of Microvascular Units The vascular topography of the tenuissimus muscle has been described by Eriksson and Myrhage (1972). Briefly, the muscle usually receives one feeding artery which enters the muscle belly in its midportion and divides into two longitudinal branches running proximally and distally as so-called central arterioles. At about every 1 mm, the central arterioles give off transversely running vessels that, after 8-14 dichotomous ramifications, give rise to a number of true capillaries, the latter being more or less parallel to the muscle fibers. The capillaries are about 1 mm long and join to form postcapillary venules. Further
VARIATIONS
OF
ENDOTHELIAL
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109
convergence leads to transverse venules which drain into a central venule parallel to the central arteriole. In this paper a “microvascular unit” (cf. Simionescu et al., 1975) begins at some of the last ramifications of the transverse arteriole and ends where a transverse venule has developed. With emphasis on wall structure, four vessel categories, designated A-D, were defined: (A) Terminal arteriolar vessels, 20-10 pm in diameter, with an incomplete muscular media or only scattered smooth muscle cells. These vessels run obliquely or transversely in the muscle and give off true capillaries. (B) Arteriolar fourth of the true capillaries, 4-7 pm in diameter. (C) Venular fourth of the true capillaries, slightly larger in diameter than B. (D) Postcapillary venules, diameter up to 30 pm. No muscular components in the incomplete media. Vessels belonging to the different categories within microvascular units were identified by a stepwise serial sectioning technique. The blocks of embedded tissue were carefully trimmed, the knife and specimen holder assembly were precisely oriented in the ultramicrotome, and 7- to lo-pm-thick sections were cut parallel to the muscle fibers. The sections were transferred to gelatin-covered glass slides. They were examined unstained in a light microscope in order to establish the connections between the microvessels and to select vessels for electron microscopy according to the criteria given above. Sections containing selected vessels were photographed in the light microscope and reembedded. Epon-filled gelatin capsules were simply put upside down over the sections on the gelatin-covered slides and the Epon was polymerized. By rinsing the glasses in hot water, the gelatin layer dissolved and the sections remained in a flat position on top of the newly polymerized Epon rods. The area of the section with identified vessels could be directly trimmed into a suitable size for ultrathin sectioning. Alternate semithin (I-pm) and ultrathin sections were cut from the original thick sections, the former being stained for light microscopy with toluidine blue. By comparison between the photographs of the thick sections, the semithin sections, and the ultrathin sections, it was possible to retrieve the selected vessels in the electron microscope. Electron Microscopic Documentntion und Morphometq Criteria were introduced concerning which endothehal cells and which parts of these in an identified vessel were to be documented for morphometric analysis. First, the endothelial cell had to exhibit distinct luminal and abluminal plasma membranes. Second, only endothelial cells with visible nuclei and clearly recognizable junctions with their neighbors were accepted. On these cells, one of the midpoints between the nucleus and the junctions was sought and centered on the viewing screen of the electron microscope before photography. Thus, the principle of selection was independent of the actual vesicle morphology. This region of the endothelial cell, the peripheral zone, seems to be the one most actively engaged in the transendothelial transport process (Simionescu et trl., 1973). The endothelial cell portions were photographed at a constant primary magnification of about 32,000~. On paper prints at a final enlargement of 96,000x, the
110
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following parameters were determined: (a) length of the luminal surface outline of the endothelial cell portion, using a cartometric wheel pen: (b) area of the transected endothelial cell cytoplasm, using a planimeter; (c) number of plasmalemmal vesicles; (d) diameter (to the nearest half-millimeter) of the vesicles including the membrane, measured parallel to the cell surface. The plasmalemmal vesicles were grouped in the following way: (1) Luminal vesicles-vesicles attached to the blood front of the cell via a neck plus vesicles without clearly discernible necks at a distance less than or equal to their own radius from the luminal plasmalemma. (2) Abluminal vesicles-vesicles with a corresponding association to the tissue front of the cell. The membrane-close vesicles without obvious necks were included in these groups since probably a large fraction of them opens on the cell surface at a plane out of the section (Karnovsky and Shea, 1970). (3) Free vesicles-vesicles in the endothelial cytoplasm situated at a distance greater than their own radius from the closest cell membrane. The examined endothelial cell portions were very attenuated, especially in the capillaries. Consequently, little space was available for vesicles to be categorized as free ones. Therefore, this group was markedly smaller than the other two. Only vesicles that were rounded and exhibited a distinct membrane were included in the counting and measuring. Section thickness was assumed to be constantly 700 8, when endothelial cell luminal surface area and cytoplasmic volume were assessed. Average thickness of endothelial cell portions was calculated as the quotient between transectional area and luminal length. Statistics All statistical analyses were performed with the Wilcoxon ranking tests. The mean value for a given variable obtained from each individual photomicrograph was used in the ranking tests. RESULTS By utilizing the stepwise serial sectioning technique, the origin, course, and end of one microvascular unit from each animal could successfully be mapped. This mapping procedure was an extremely time-consuming task. In numerous cases the course and connections of the microvessels were so intricate that they were impossible to follow. The main problem, which is impossible to illustrate in this communication, was connected with the meandering course in a direction perpendicular to the plane of sectioning of especially the arteriolar and precapillary vessels. The postcapillary venules with their affluent capillaries were, on the other hand, quite easily identified, since they were generally ordered in planes parallel to the sections, that is, parallel to the surface of the attenuated muscle. In order to ensure a reasonably equal preservation of the vessel segments to be analyzed, only vessels situated in the superficial 50 pm layer of the muscles were documented. There were no obvious signs of thrombus formations, bleedings, or inflammatory reactions in the tissues.
VARIATIONS
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111
With the adopted selection criteria, it was possible to obtain S-12 photomicrographs from each of the above-defined vessel categories in each animal. Individual endothelial cells were recorded only once. The final material comprised 191 prints. For a section thickness of 700 A, the included endothelial cell portions covered a total luminal vessel surface area of about 27.6 pm2 and occupied an aggregate volume of about 6.7pm3. The average endothelial cell thickness was in the terminal arterioles 0.25 pm, in the arteriolar end of the capillaries 0.22 pm, in the venular end of the capillaries 0.23pm, and in the postcapillary venules 0.35 pm. In all, 1164 luminal vesicles, 1659 abluminal vesicles, and 374 free vesicles were counted and measured. A survey of the size distribution of the plasmalemmal vesicles in the whole material is given in Table 1. It is seen that most vesicle diameters ranged between 675 and 835 A, the peak frequency lying around 780 A. The interest was focused on the variations in numbers, distributions, and sizes of membrane-attached vesicles along the consecutive segments of the individual microvascular units. This analysis is accounted for in Figs. l-5, where each figure represents one microvascular unit, i.e., one animal. The “a” figures show the mean luminal and abluminal vesicle counts per square micrometer luminal vessel surface area in the different vessel categories, and the “b” figures show the mean diameters of the plasmalemma-associated vesicles within the vessel segments. Vesicle Frequencies
per Squcrre Micrometer
Luminal
Vessel Surface Area
In all microvascular segments except three, the abluminal vesicles outnumbered the luminal ones. This is a conspicuous feature of all “a” figures, although the differences were not regularly statistically significant. Equal or reversed mean frequencies of the attached vesicles were only encountered in animal 1, vessel category A, and in animal 4, vessel categories A and B. The comparisons of the consecutive vascular segments showed that in animal 1 the precapillary vessels had the minimal frequency of vesicles and continuously increasing numbers toward the venular vessels. In animals 2 and 3, on the other hand, the luminal and abluminal vesicles were significantly fewer in the postcapillary vessels. Note that in animal 3 the profile formed by the bars in the figure is practically opposite to that in animal 1. Animals 4 and 5 displayed less gradientlike distributions of the vesicles, although at markedly different numerical levels. Vesicle Diameters (Figs. lb-5b) Generally, the vesicle size was not significantly different between the two aspects of the endothelium. It is remarkable, though, that in vessel categories A and B the mean diameter of the luminal vesicles was slightly larger than that of the abluminal vesicles in 9 cases out of 10, while in categories C and D the abluminal vesicles were the larger ones in 7 cases out of 10. The longitudinal comparisons indicated that in animals 1, 2, and 5 the abluminal vesicles of vessel category D were larger than those of the precapillary vessels and those of some capillary segments. Quite a different size distribution of the vesicles
15
Total
59
2 6 1 10 9 0 9 13 0 1 6 2
575 (5.5)
(6)
178
408
31 49 7 46 63 12 51 66 15 22 38 8
675
(6.5)
625 12 17 5 20 28 6 27 36 5 10 11 1
1
IN THE DIFFERENT
TABLE
668
67 79 20 54 112 21 69 112 20 48 52 14
730 (7)
687
61 83 15 74 115 17 64 98 27 48 70 15
780 (7.5)
573
57 64 13 56 87 16 48 87 18 39 73 15
(8)
835
340
35 33 7 37 41 10 29 44 9 29 34 32
(8.3
885
Vesicle diameter, A (mm in prints)
OF VESICLES
162
20 22 5 13 15 7 14 21 4 11 25 5
940 (9)
VESSEL
78
11 5 3 10 5 2 7 6 3 7 11 8
990 (9.5)
CATEGORIES"
29
5 5 0 0 0 2 2 4 0 2 6 3
1040 (10)
3197
301 366 76 322 475 93 322 492 102 219 326 103
Total
27.64
6.48
7.29
7.26
6.61
Total luminal surface area (me
11 44 65 13 44 67 14 34 50 16
46 55
Number of vesicles /NJ2
,J The vesicle diameter (membrane included) was measured to the closest half-millimeter on the prints. The angstrom notation in the column heading over each size class was calculated from the millimeter value (in parentheses) assuming a constant linear magnification of %,000x in the prints. Each column thus represents the number of vesicles within a range of sizes, the midpoint of which is denoted by the angstrom value. Endothelial luminal surface area estimated for a section thickness of 700 A.
D
C
B
0 3 0 2 0 0 2 5 I 2 0 0
Luminal Abluminal Free Luminal Abluminal Free Luminal Abluminal Free Luminal Abluminal Free
A
520 (5)
Vesicle type
Vessel category
SIZE DISTRIBUTION
E z 7 7 a F z Ls %
VARIATIONS
OF
ENDOTHELIAL
VESICULATION
lb
18 900
I*
850
800
B Y sb
AA !I”.t C
850
,1 k
b
! . b
”
B
c
I”
.b cl
113
114
BENGT
R.
JOHANSSON 3b
900
660
606
760
700
I” .b A
Ill * 6
II ” .b c
” .I D
4b SDC
660
I!i-l SD0
t
l
*
760
9
”
.b
C
I”
.b
D
700
VARIATIONS
OF
ENDOTHELIAL
115
VESICULATION
Sb
900 *
i;
, 1
960
900
760
m
b
C
7Da
D
A
I” .b B
C
D
FIGS. 1-5. Frequency (a) and diameters (b) of luminal and abluminal vesicles in the four vessel categories. Each figure represents one microvascular unit (one animal): each bar represents the mean value (?SE) of measurements in 8-12 photomicrographs. Results of statistical tests are shown by symbols on the bars: An asterisk denotes a statistically demonstrable difference between values within a vessel category; capital letters denote differences between the vessel categories. Significance level: symbols without parentheses P < 0.05; symbols within parentheses 0.1 > P > 0.05. In the longitudinal tests the luminal and abluminal vesicle subpopulations were compared separately and not with each other, i.e., a capital letter on a luminal vesicle bar denotes a difference of the luminal vesicles only between the two vessel categories.
was found in animal 4, where the luminal vesicles in the terminal arterioles were significantly larger than in other locations. A markedly even size distribution of both vesicle types was recorded along the microvascular unit of animal 3. DISCUSSION Ultrastructural studies have shown that the abundant plasmalemmal vesicles in the endothelium of continuous blood capillaries are involved in a transendothelial transport of macromolecules (Karnovsky, 1967; Bruns and Palade, 1968b; Simionescu et al., 1973). Several investigators (Mayerson et rrl., 1960; Renkin, 1964; Bruns and Palade, 1968b) have suggested that the vesicles are the structural counterpart of the capillary large pore system, permeable to macromolecules (Grotte, 1956). The vesicular transport is a two-way process (Johansson, 197%). The net vesicle-mediated flux of macromolecules across the endothelium is accordingly influenced by the quantitative relationship of the vesicular transports in
116
BENGT
R. JOHANSSON
the respective directions. An indirect measure of the prevailing transport activity should be obtained by assessing in the electron microscope the frequency, distribution, and size of the vesicles. Some authors have pointed out that in capillaries the abluminally attached vesicles are regularly more numerous than the luminal ones (Bruns and Palade, 1968a; Simionescu ef al., 1974; Johansson, 1979b). The present results were in full accord with these observations. However, other workers deny such a difference between the two aspects of the endothelium (Leak, 1971; Casley-Smith et al., 1975). In a previous study, I was able to demonstrate that the predominance of abluminal vesicles was reduced after elevation of the capillary hydrostatic pressure (Johansson, 1979b). It was therefore suggested that the balance of the vesicle-mediated transports in the respective direction was variable as a response to variations in hemodynamic parameters (Johansson, 1979b). Moreover, in a preliminary study I obtained evidence that the mean size of the abluminal vesicles was also reduced upon elevation of the capillary hydrostatic pressure (Johansson, 1976). Against this background the present study was undertaken in order to elucidate whether or not systematic differences in the endothelial vesiculation could be demonstrated along the consecutive segments of nutritive vessels belonging to individual microvascular units. Considering the above-mentioned differences in vesicle distribution and size at different levels of capillary hydrostatic pressure (Johansson, 1979b), it appeared plausible that the differences in hemodynamics and microenvironments along the arteriole-capillary-venule sequence should be reflected in ordered variations of vesicle numbers, distributions, and sizes. However, this idea was not corroborated by the observations, which showed fundamental differences between the animals and no recognizable pattern of vesiculation in the various vessel segments. Of course, the results obtained could be ascribed to an inadequate technique, in the first place, unsatisfactory fixation. However, since the vessels included within a microvascular unit were located in a small volume of tissue, it is improbable that the recorded differences in vesiculation were caused artifactually by uneven fixation. Moreover, the smooth, gradient-like profile of several of the histograms (Figs. l-5) makes it unlikely that the differences, some of which were statistically significant, were merely incidental. Consequently, it seems justified to conclude that the different patterns of vesiculation were due to different conditions within or outside the vessel segments at the time of fixation. Although the nature of these differences is not known, the observations indicate that the microvascular endothelium is reactive to more factors than the intraluminal hydrostatic pressure, as described in the preceding paper (Johansson, 1979b). This possibility merits further systematic analysis. REFERENCES BRANEMARK, P. -I., AND ERIKSSON, E. (1972). Method for studying qualitative and quantitative changes of blood flow in skeletal muscle. Acta Physiol. &and. 84, 284-288. BRUNS, R. R., AND PALADE, G. E. (1%8a). Studies on blood capillaries. I. General organisation blood capillaries in muscle. J. Cell Biol. 37, 244-276
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BRUNS, R. R., AND PALADE, G. E. (1%8b). Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J. Ce// Bid/. 37, 277-299. CASLEY-SMITH, J. R. (1971). Endothelial fenestrae in intestinal villi: Differences between the arterial and venous ends of the capillaries. Microvasc. Res. 3, 49-68. CASLEY-SMITH, J. R., GREEN, H. S., HARRIS, J. L., AND WADEY, P. I. (1975). The quantitative morphology of skeletal muscle capillaries in relation to permeability. Microvasc. Res. 10, 43-64. ERIKSSON, E., AND MYRHAGE, R. (1972). Microvascular dimensions and blood flow in skeletal muscle. Actu Physiol. Stand. 86, 211-222. FRONEK, K., AND ZWEIFACH, B. W. (1975). Microvascular pressure distribution in skeletal muscle and the effect of vasodilation. Amer. J. Physiol. 228, 791-796. GROTTE, G. (1956). Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Stand. (Suppl.) 211, l-84. JOHANSSON, B. R. (1976). Capillary pressure and endothelial cell ultrastructure. Bib/. Anat. 15, 528-530. JOHANSSON, B. R. (1978). Permeability of muscle capillaries to interstitially microinjected horseradish peroxidase. Microvasc. Res. 16, 340-353. JOHANSSON, B. R. (1979). Quantitative ultrastructural morphometry of blood capillary endothelium in skeletal muscle. Effect of venous pressure. Microvnsc. Res. 17, 118-130. KARNOVSKY, M. J. (1967). The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236. KARNOVSKY, M. J., AND SHEA, S. M. (1970). Transcapillary transport by pinocytosis. Microvusc. Res. 2, 353-360. LEAK, L. V. (1971). Frozen-fractured images of blood capillaries in heart tissue. J. Ultrustruct. Res. 35, 127-146. MAYERSON, H. S., WOLFRAM, C. G., SHIRLEY, H. H., JR., AND WASSERMAN, K. (1960). Regional differences in capillary permeability. Amer. 1. Physiol. 198, 155-160. RENKIN, E. M. (1964). Transport of large molecules across capillary walls. Physiologist 7, 13-28. RHODIN, J. A. G. (1967). The ultrastructure of mammalian arterioles and precapillary sphincters. J. Ultrastruct. Res. 18, 181-223. RHODIN, J. A. G. (1968). Ultrastructure of mammalian venous capillaries, venules and small collecting veins. J. lJltra.struct. Res. 25, 452-500. SIMIONESCU, N., SIMIONESCU, M., AND PALADE. G. E. (1973). Permeability of muscle capillaries to exogenous myoglobin. .I. Cell Biol. 57, 424-452. SIMIONESCU, M., SIMIONESCU, N., AND PALADE, G. E. (1974). Morphometric data on the endothelium of blood capillaries. J. Cell Biol. 60, 128-152. SIMIONESCU, M., SIMIONESCU, N., AND PALADE, G. E. (1975). Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature. J. Cell Biol. 67, 863-885. SIMIONESCU, N., SIMIONESCU, M., AND PALADE, G. E. (1976). Structural basis of permeability in sequential segments of the microvasculature. J. Cell Biol. 70, 186a.
RESEARCH
17, 107-117
(1979)
Size and Distribution of Endothelial Plasmalemmal Vesicles in Consecutive Segments of the Microvasculature in Cat Skeletal Muscle BENCT R. JOHANSSON Department
of Anatomy,
University of Gtiteborg,
Giiteborg,
Sweden
Received September II, 1977 Vessels belonging to five individual microvascular units (arteriole-capillary-venule sequence) in the tenuissimus muscle of cat were identified in the electron microscope by utilizing a serial sectioning technique. Morphometric analyses were performed on the plasmalemmal vesicle population in four defined vessel segments: terminal arterioles, arteriolar fourth of capillaries, venular fourth of capillaries, and postcapillary venules. The sizes and numbers of vesicles, classified as luminal, abluminal, and free vesicles, were assessed. The abluminal vesicles were regularly more numerous than the luminal ones in the same segment. There were markedly different patterns of vesiculation along the five microvascular units. This finding was interpreted tentatively as indicating a reactivity of the endothelial transport function to factors of an unknown nature in the local microenvironment.
INTRODUCTION Ultrastructural studies on the microvasculature are generally performed by collecting data from not thoroughly characterized parts of the nutritive vessels. The concept “capillary” becomes a common designation for small vessels with a maximal diameter, and their location in the terminal arteriole-true capillarypostcapillary venule sequence is often uncertain. However, longitudinal, functional,. and structural differentiations do exist along the consecutive segments of the nutritive vessel. These differentiations have been detailed in intestinal villi (Casley-Smith, 1971) and in tissues with a limited extension in the third dimension both with conventional electron microscopy (Rhodin, 1967, 1968) and with freeze-fracturing techniques (Simionescu et ol., 1975). Recently, Simionescu et al. (1976) also reported variations in the structural substrate of vascular permeability in sequential segments of the microvasculature in mouse diaphragm. Their findings indicated, for example, that the rate of transendothelial transport via plasmalemmal vesicles increased toward the venular end of the capillaries (Simionescu et al., 1976). In two preceding investigations on rat skeletal muscle, I was able to demonstrate that the number, size, and distribution of endothelial plasmalemmal vesicles were different at different capillary hydrostatic pressures (Johansson, 1976,1979b). These observations thus indicated the possibility of a coupling between vesicle morphology and intravascular pressure level. The aim of the present study was to elucidate with the electron microscope whether ultrastructural differentiations exist in the endothelium, notably the plasmalemmal vesicle system, along the different microvascular segments in 107
‘X26-2862i79~020107-I 1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
108
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skeletal muscle. It was decided not to collect randomly selected vessels of defined sizes but to compare the endothelial ultrastructure of vascular segments belonging to the same microvascular unit derived from a common arteriole and ending up in a common venule. Thus, also the extremities of the capillaries were defined and compared. MATERIALS
AND
METHODS
Animals and Specimen Excision The study was performed on the tenuissimus muscle in cats. This muscle has been utilized in intravital microscopic studies of the microcirculation (Eriksson and Myrhage, 1972; Fronek and Zweifach, 1975). Five cats, body weight 2-2.5 kg, were used. The animals were anesthetized with chloralose, 50 mg/kg body weight, and were breathing spontaneously. The left tenuissimus muscle was exposed surgically according to Brinemark and Eriksson (1972). After exposing the muscle, its midportion was grasped with a clamp and simultaneously flooded with fixative. To one shank of the clamp, a 1.5 x 2-cm glass plate was adapted; to the other, a slightly smaller metallic frame. The use of a clamp allowed the muscle to be kept flat and undistorted throughout prefixation, which facilitated the subsequent identification of vascular segments. Moreover, the retained red blood cells in the vessels served as “markers” in the different steps of vessel identification. After in situ fixation for 5 min, the muscle was cut outside the frame of the clamp and the whole assembly was immersed in cold fixative for at least 2 hr. Tissue Processing and Electron Microscopy The fixative was a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.09 M Na cacodylate buffer, pH 7.2. After rinsing in fresh buffer, the midportions of the muscles were cut transversely into five to seven approximately 1.5-mm-long pieces which were postfixed in 1% 0~0, in 0.1 M Na cacodylate buffer, pH 7.2, for 2 hr. Dehydration in graded series of ethanol and embedding in Epon in flat molds were performed according to routine procedures. For all sectioning, an LKB Ultrotome with glass knives was used. Ultrathin sections were mounted on copper grids and contrasted with lead citrate and uranyl acetate. Only sections with a silver-gray interference color were collected. The sections were examined in a Philips EM 300 electron microscope. Its magnification was calibrated with a carbon grating replica, 2160 lines/mm. Identification of Microvascular Units The vascular topography of the tenuissimus muscle has been described by Eriksson and Myrhage (1972). Briefly, the muscle usually receives one feeding artery which enters the muscle belly in its midportion and divides into two longitudinal branches running proximally and distally as so-called central arterioles. At about every 1 mm, the central arterioles give off transversely running vessels that, after 8-14 dichotomous ramifications, give rise to a number of true capillaries, the latter being more or less parallel to the muscle fibers. The capillaries are about 1 mm long and join to form postcapillary venules. Further
VARIATIONS
OF
ENDOTHELIAL
VESICULATION
109
convergence leads to transverse venules which drain into a central venule parallel to the central arteriole. In this paper a “microvascular unit” (cf. Simionescu et al., 1975) begins at some of the last ramifications of the transverse arteriole and ends where a transverse venule has developed. With emphasis on wall structure, four vessel categories, designated A-D, were defined: (A) Terminal arteriolar vessels, 20-10 pm in diameter, with an incomplete muscular media or only scattered smooth muscle cells. These vessels run obliquely or transversely in the muscle and give off true capillaries. (B) Arteriolar fourth of the true capillaries, 4-7 pm in diameter. (C) Venular fourth of the true capillaries, slightly larger in diameter than B. (D) Postcapillary venules, diameter up to 30 pm. No muscular components in the incomplete media. Vessels belonging to the different categories within microvascular units were identified by a stepwise serial sectioning technique. The blocks of embedded tissue were carefully trimmed, the knife and specimen holder assembly were precisely oriented in the ultramicrotome, and 7- to lo-pm-thick sections were cut parallel to the muscle fibers. The sections were transferred to gelatin-covered glass slides. They were examined unstained in a light microscope in order to establish the connections between the microvessels and to select vessels for electron microscopy according to the criteria given above. Sections containing selected vessels were photographed in the light microscope and reembedded. Epon-filled gelatin capsules were simply put upside down over the sections on the gelatin-covered slides and the Epon was polymerized. By rinsing the glasses in hot water, the gelatin layer dissolved and the sections remained in a flat position on top of the newly polymerized Epon rods. The area of the section with identified vessels could be directly trimmed into a suitable size for ultrathin sectioning. Alternate semithin (I-pm) and ultrathin sections were cut from the original thick sections, the former being stained for light microscopy with toluidine blue. By comparison between the photographs of the thick sections, the semithin sections, and the ultrathin sections, it was possible to retrieve the selected vessels in the electron microscope. Electron Microscopic Documentntion und Morphometq Criteria were introduced concerning which endothehal cells and which parts of these in an identified vessel were to be documented for morphometric analysis. First, the endothelial cell had to exhibit distinct luminal and abluminal plasma membranes. Second, only endothelial cells with visible nuclei and clearly recognizable junctions with their neighbors were accepted. On these cells, one of the midpoints between the nucleus and the junctions was sought and centered on the viewing screen of the electron microscope before photography. Thus, the principle of selection was independent of the actual vesicle morphology. This region of the endothelial cell, the peripheral zone, seems to be the one most actively engaged in the transendothelial transport process (Simionescu et trl., 1973). The endothelial cell portions were photographed at a constant primary magnification of about 32,000~. On paper prints at a final enlargement of 96,000x, the
110
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following parameters were determined: (a) length of the luminal surface outline of the endothelial cell portion, using a cartometric wheel pen: (b) area of the transected endothelial cell cytoplasm, using a planimeter; (c) number of plasmalemmal vesicles; (d) diameter (to the nearest half-millimeter) of the vesicles including the membrane, measured parallel to the cell surface. The plasmalemmal vesicles were grouped in the following way: (1) Luminal vesicles-vesicles attached to the blood front of the cell via a neck plus vesicles without clearly discernible necks at a distance less than or equal to their own radius from the luminal plasmalemma. (2) Abluminal vesicles-vesicles with a corresponding association to the tissue front of the cell. The membrane-close vesicles without obvious necks were included in these groups since probably a large fraction of them opens on the cell surface at a plane out of the section (Karnovsky and Shea, 1970). (3) Free vesicles-vesicles in the endothelial cytoplasm situated at a distance greater than their own radius from the closest cell membrane. The examined endothelial cell portions were very attenuated, especially in the capillaries. Consequently, little space was available for vesicles to be categorized as free ones. Therefore, this group was markedly smaller than the other two. Only vesicles that were rounded and exhibited a distinct membrane were included in the counting and measuring. Section thickness was assumed to be constantly 700 8, when endothelial cell luminal surface area and cytoplasmic volume were assessed. Average thickness of endothelial cell portions was calculated as the quotient between transectional area and luminal length. Statistics All statistical analyses were performed with the Wilcoxon ranking tests. The mean value for a given variable obtained from each individual photomicrograph was used in the ranking tests. RESULTS By utilizing the stepwise serial sectioning technique, the origin, course, and end of one microvascular unit from each animal could successfully be mapped. This mapping procedure was an extremely time-consuming task. In numerous cases the course and connections of the microvessels were so intricate that they were impossible to follow. The main problem, which is impossible to illustrate in this communication, was connected with the meandering course in a direction perpendicular to the plane of sectioning of especially the arteriolar and precapillary vessels. The postcapillary venules with their affluent capillaries were, on the other hand, quite easily identified, since they were generally ordered in planes parallel to the sections, that is, parallel to the surface of the attenuated muscle. In order to ensure a reasonably equal preservation of the vessel segments to be analyzed, only vessels situated in the superficial 50 pm layer of the muscles were documented. There were no obvious signs of thrombus formations, bleedings, or inflammatory reactions in the tissues.
VARIATIONS
OF
ENDOTHELIAL
VESICULATION
111
With the adopted selection criteria, it was possible to obtain S-12 photomicrographs from each of the above-defined vessel categories in each animal. Individual endothelial cells were recorded only once. The final material comprised 191 prints. For a section thickness of 700 A, the included endothelial cell portions covered a total luminal vessel surface area of about 27.6 pm2 and occupied an aggregate volume of about 6.7pm3. The average endothelial cell thickness was in the terminal arterioles 0.25 pm, in the arteriolar end of the capillaries 0.22 pm, in the venular end of the capillaries 0.23pm, and in the postcapillary venules 0.35 pm. In all, 1164 luminal vesicles, 1659 abluminal vesicles, and 374 free vesicles were counted and measured. A survey of the size distribution of the plasmalemmal vesicles in the whole material is given in Table 1. It is seen that most vesicle diameters ranged between 675 and 835 A, the peak frequency lying around 780 A. The interest was focused on the variations in numbers, distributions, and sizes of membrane-attached vesicles along the consecutive segments of the individual microvascular units. This analysis is accounted for in Figs. l-5, where each figure represents one microvascular unit, i.e., one animal. The “a” figures show the mean luminal and abluminal vesicle counts per square micrometer luminal vessel surface area in the different vessel categories, and the “b” figures show the mean diameters of the plasmalemma-associated vesicles within the vessel segments. Vesicle Frequencies
per Squcrre Micrometer
Luminal
Vessel Surface Area
In all microvascular segments except three, the abluminal vesicles outnumbered the luminal ones. This is a conspicuous feature of all “a” figures, although the differences were not regularly statistically significant. Equal or reversed mean frequencies of the attached vesicles were only encountered in animal 1, vessel category A, and in animal 4, vessel categories A and B. The comparisons of the consecutive vascular segments showed that in animal 1 the precapillary vessels had the minimal frequency of vesicles and continuously increasing numbers toward the venular vessels. In animals 2 and 3, on the other hand, the luminal and abluminal vesicles were significantly fewer in the postcapillary vessels. Note that in animal 3 the profile formed by the bars in the figure is practically opposite to that in animal 1. Animals 4 and 5 displayed less gradientlike distributions of the vesicles, although at markedly different numerical levels. Vesicle Diameters (Figs. lb-5b) Generally, the vesicle size was not significantly different between the two aspects of the endothelium. It is remarkable, though, that in vessel categories A and B the mean diameter of the luminal vesicles was slightly larger than that of the abluminal vesicles in 9 cases out of 10, while in categories C and D the abluminal vesicles were the larger ones in 7 cases out of 10. The longitudinal comparisons indicated that in animals 1, 2, and 5 the abluminal vesicles of vessel category D were larger than those of the precapillary vessels and those of some capillary segments. Quite a different size distribution of the vesicles
15
Total
59
2 6 1 10 9 0 9 13 0 1 6 2
575 (5.5)
(6)
178
408
31 49 7 46 63 12 51 66 15 22 38 8
675
(6.5)
625 12 17 5 20 28 6 27 36 5 10 11 1
1
IN THE DIFFERENT
TABLE
668
67 79 20 54 112 21 69 112 20 48 52 14
730 (7)
687
61 83 15 74 115 17 64 98 27 48 70 15
780 (7.5)
573
57 64 13 56 87 16 48 87 18 39 73 15
(8)
835
340
35 33 7 37 41 10 29 44 9 29 34 32
(8.3
885
Vesicle diameter, A (mm in prints)
OF VESICLES
162
20 22 5 13 15 7 14 21 4 11 25 5
940 (9)
VESSEL
78
11 5 3 10 5 2 7 6 3 7 11 8
990 (9.5)
CATEGORIES"
29
5 5 0 0 0 2 2 4 0 2 6 3
1040 (10)
3197
301 366 76 322 475 93 322 492 102 219 326 103
Total
27.64
6.48
7.29
7.26
6.61
Total luminal surface area (me
11 44 65 13 44 67 14 34 50 16
46 55
Number of vesicles /NJ2
,J The vesicle diameter (membrane included) was measured to the closest half-millimeter on the prints. The angstrom notation in the column heading over each size class was calculated from the millimeter value (in parentheses) assuming a constant linear magnification of %,000x in the prints. Each column thus represents the number of vesicles within a range of sizes, the midpoint of which is denoted by the angstrom value. Endothelial luminal surface area estimated for a section thickness of 700 A.
D
C
B
0 3 0 2 0 0 2 5 I 2 0 0
Luminal Abluminal Free Luminal Abluminal Free Luminal Abluminal Free Luminal Abluminal Free
A
520 (5)
Vesicle type
Vessel category
SIZE DISTRIBUTION
E z 7 7 a F z Ls %
VARIATIONS
OF
ENDOTHELIAL
VESICULATION
lb
18 900
I*
850
800
B Y sb
AA !I”.t C
850
,1 k
b
! . b
”
B
c
I”
.b cl
113
114
BENGT
R.
JOHANSSON 3b
900
660
606
760
700
I” .b A
Ill * 6
II ” .b c
” .I D
4b SDC
660
I!i-l SD0
t
l
*
760
9
”
.b
C
I”
.b
D
700
VARIATIONS
OF
ENDOTHELIAL
115
VESICULATION
Sb
900 *
i;
, 1
960
900
760
m
b
C
7Da
D
A
I” .b B
C
D
FIGS. 1-5. Frequency (a) and diameters (b) of luminal and abluminal vesicles in the four vessel categories. Each figure represents one microvascular unit (one animal): each bar represents the mean value (?SE) of measurements in 8-12 photomicrographs. Results of statistical tests are shown by symbols on the bars: An asterisk denotes a statistically demonstrable difference between values within a vessel category; capital letters denote differences between the vessel categories. Significance level: symbols without parentheses P < 0.05; symbols within parentheses 0.1 > P > 0.05. In the longitudinal tests the luminal and abluminal vesicle subpopulations were compared separately and not with each other, i.e., a capital letter on a luminal vesicle bar denotes a difference of the luminal vesicles only between the two vessel categories.
was found in animal 4, where the luminal vesicles in the terminal arterioles were significantly larger than in other locations. A markedly even size distribution of both vesicle types was recorded along the microvascular unit of animal 3. DISCUSSION Ultrastructural studies have shown that the abundant plasmalemmal vesicles in the endothelium of continuous blood capillaries are involved in a transendothelial transport of macromolecules (Karnovsky, 1967; Bruns and Palade, 1968b; Simionescu et al., 1973). Several investigators (Mayerson et rrl., 1960; Renkin, 1964; Bruns and Palade, 1968b) have suggested that the vesicles are the structural counterpart of the capillary large pore system, permeable to macromolecules (Grotte, 1956). The vesicular transport is a two-way process (Johansson, 197%). The net vesicle-mediated flux of macromolecules across the endothelium is accordingly influenced by the quantitative relationship of the vesicular transports in
116
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R. JOHANSSON
the respective directions. An indirect measure of the prevailing transport activity should be obtained by assessing in the electron microscope the frequency, distribution, and size of the vesicles. Some authors have pointed out that in capillaries the abluminally attached vesicles are regularly more numerous than the luminal ones (Bruns and Palade, 1968a; Simionescu ef al., 1974; Johansson, 1979b). The present results were in full accord with these observations. However, other workers deny such a difference between the two aspects of the endothelium (Leak, 1971; Casley-Smith et al., 1975). In a previous study, I was able to demonstrate that the predominance of abluminal vesicles was reduced after elevation of the capillary hydrostatic pressure (Johansson, 1979b). It was therefore suggested that the balance of the vesicle-mediated transports in the respective direction was variable as a response to variations in hemodynamic parameters (Johansson, 1979b). Moreover, in a preliminary study I obtained evidence that the mean size of the abluminal vesicles was also reduced upon elevation of the capillary hydrostatic pressure (Johansson, 1976). Against this background the present study was undertaken in order to elucidate whether or not systematic differences in the endothelial vesiculation could be demonstrated along the consecutive segments of nutritive vessels belonging to individual microvascular units. Considering the above-mentioned differences in vesicle distribution and size at different levels of capillary hydrostatic pressure (Johansson, 1979b), it appeared plausible that the differences in hemodynamics and microenvironments along the arteriole-capillary-venule sequence should be reflected in ordered variations of vesicle numbers, distributions, and sizes. However, this idea was not corroborated by the observations, which showed fundamental differences between the animals and no recognizable pattern of vesiculation in the various vessel segments. Of course, the results obtained could be ascribed to an inadequate technique, in the first place, unsatisfactory fixation. However, since the vessels included within a microvascular unit were located in a small volume of tissue, it is improbable that the recorded differences in vesiculation were caused artifactually by uneven fixation. Moreover, the smooth, gradient-like profile of several of the histograms (Figs. l-5) makes it unlikely that the differences, some of which were statistically significant, were merely incidental. Consequently, it seems justified to conclude that the different patterns of vesiculation were due to different conditions within or outside the vessel segments at the time of fixation. Although the nature of these differences is not known, the observations indicate that the microvascular endothelium is reactive to more factors than the intraluminal hydrostatic pressure, as described in the preceding paper (Johansson, 1979b). This possibility merits further systematic analysis. REFERENCES BRANEMARK, P. -I., AND ERIKSSON, E. (1972). Method for studying qualitative and quantitative changes of blood flow in skeletal muscle. Acta Physiol. &and. 84, 284-288. BRUNS, R. R., AND PALADE, G. E. (1%8a). Studies on blood capillaries. I. General organisation blood capillaries in muscle. J. Cell Biol. 37, 244-276
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VESICULATION
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BRUNS, R. R., AND PALADE, G. E. (1%8b). Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J. Ce// Bid/. 37, 277-299. CASLEY-SMITH, J. R. (1971). Endothelial fenestrae in intestinal villi: Differences between the arterial and venous ends of the capillaries. Microvasc. Res. 3, 49-68. CASLEY-SMITH, J. R., GREEN, H. S., HARRIS, J. L., AND WADEY, P. I. (1975). The quantitative morphology of skeletal muscle capillaries in relation to permeability. Microvasc. Res. 10, 43-64. ERIKSSON, E., AND MYRHAGE, R. (1972). Microvascular dimensions and blood flow in skeletal muscle. Actu Physiol. Stand. 86, 211-222. FRONEK, K., AND ZWEIFACH, B. W. (1975). Microvascular pressure distribution in skeletal muscle and the effect of vasodilation. Amer. J. Physiol. 228, 791-796. GROTTE, G. (1956). Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Stand. (Suppl.) 211, l-84. JOHANSSON, B. R. (1976). Capillary pressure and endothelial cell ultrastructure. Bib/. Anat. 15, 528-530. JOHANSSON, B. R. (1978). Permeability of muscle capillaries to interstitially microinjected horseradish peroxidase. Microvasc. Res. 16, 340-353. JOHANSSON, B. R. (1979). Quantitative ultrastructural morphometry of blood capillary endothelium in skeletal muscle. Effect of venous pressure. Microvnsc. Res. 17, 118-130. KARNOVSKY, M. J. (1967). The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236. KARNOVSKY, M. J., AND SHEA, S. M. (1970). Transcapillary transport by pinocytosis. Microvusc. Res. 2, 353-360. LEAK, L. V. (1971). Frozen-fractured images of blood capillaries in heart tissue. J. Ultrustruct. Res. 35, 127-146. MAYERSON, H. S., WOLFRAM, C. G., SHIRLEY, H. H., JR., AND WASSERMAN, K. (1960). Regional differences in capillary permeability. Amer. 1. Physiol. 198, 155-160. RENKIN, E. M. (1964). Transport of large molecules across capillary walls. Physiologist 7, 13-28. RHODIN, J. A. G. (1967). The ultrastructure of mammalian arterioles and precapillary sphincters. J. Ultrastruct. Res. 18, 181-223. RHODIN, J. A. G. (1968). Ultrastructure of mammalian venous capillaries, venules and small collecting veins. J. lJltra.struct. Res. 25, 452-500. SIMIONESCU, N., SIMIONESCU, M., AND PALADE. G. E. (1973). Permeability of muscle capillaries to exogenous myoglobin. .I. Cell Biol. 57, 424-452. SIMIONESCU, M., SIMIONESCU, N., AND PALADE, G. E. (1974). Morphometric data on the endothelium of blood capillaries. J. Cell Biol. 60, 128-152. SIMIONESCU, M., SIMIONESCU, N., AND PALADE, G. E. (1975). Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature. J. Cell Biol. 67, 863-885. SIMIONESCU, N., SIMIONESCU, M., AND PALADE, G. E. (1976). Structural basis of permeability in sequential segments of the microvasculature. J. Cell Biol. 70, 186a.