EXPERIMENTAL
AND
Regional
XfOLECULAR
PATHOLOGY
24, 23-34
( 19%)
Variation in Rat Aortic Endothelial Surface Relationship to Regional Aortic Permeability ~IC~~~.KATO~A~T~ODO~
Biology
~epe~~~,
Received
208 Life University April
M.HOLL~S
Sceinces I, The Pen~~~l~~~~i~ Pu&, Penns~~vaniu 16802
28, 1975,
morphology:
and
in revised
fom
June
State
Uniuersit~,
9, 1975
The regional variation of rat aortic lumenal surface topography has been examined by employing techniques of scanning electron microscopy. This study was undertaken in an attempt to gain additional insight into the current controversy regarding endothelial folds and cross bridges and their possible role with respect to regulation of vessel transmural permeability. Within the ascending aorta, aortic arch, and upper third of the thoracic aorta distinct, prominent endothelial folds connected by interlinking cross bridges were easily identifiable. The middle third of the thoracic aorta contained less prominent endothelial folds; photomicrographs from these regions consistently failed to reveal the presence of any interfold cross bridges. In the lower third of the thoracic aorta, endothelial folds were notably lacking. These differences in regional aortic surface topography have been discussed in light of known regional differences in aortic wall permeability and with respect to regional differences in appearance of endothelial silver-staining lines.
INTRODUCTION Through use of vital dyes, fluorescent markers, and radioisotopes it is now generally recognized that regional variations in aortic pe~eabili~ exist within most mammalian species (Duncan et al., 1959, 196I, 1962; J$rgensen et al., 1970; Klynstra and Biittcher, 1970; Katora and Hollis, 1975). Regions of increased permeability include such areas as the inner curvatnre of the aortic arch, sinuses of Valsalva, orifices of vessels to the head and neck, and thoracic aortic intercostal ostea (Friedman and Byers, 1963; Packham et at., 1967; Klynstra and Bottcher, 1970; Jpirgensenet al., 1972; Giacomelli and Wiener, 1974). In numerous studies, among which are those of Fry ( 1969a,b, 1973), these areas have been identified by increased uptake of Evans blue dye and coincide with regions in which hemodynamic stressesare locally elevated. It has been proposed that these regions represent areas in which hemodynamic-mediated endothelial injury has occurred. Aortic regions showing focal blueing are associated with increased uptake of fibrinogen (Bell et al., 1974b), albumin (Packham et al., 1967; Bell et al., 1974a), aud unesterified cholesterol (Somer and Schwartz, 1972); such regions also show increased endothelial turnover ( Caplan and Schwartz, 1973). Recently, Caplan et al. (1974) h ave described differences in thickness and the number of gaps * Supported
by
NSF
Grant
GR-38136.
Send
reprint 23
Copyright All tights
1976 by Academic Press, Inc. reproduction in any form reserved.
requests
to Dr.
Theodore
M.
Hollis.
KATORA
24
AND HOLLIS
located within silver staining lines of Haiitchen preparations of endothelium obtained from areas of aortic blueing and nonblueing in an attempt to determine whether morphological differences in these lines (intercellular junctions) can account for this altered aortic transmural permeability. In a series of scanning electron microscopy (SEM) studies, Shimamoto and co-workers (1969a) have suggested that endothelial intercellular cross bridges are vital and active components in the regulation of aortic endothelial permeability. Since the endothelium is now recognized as occupying a central role in regulation of aortic transmural permeability, and in light of these previous studies (Shimamoto et al., 1969a,b; Giacomelli and Wiener, 1974; Caplan et al., 1974), it would seem logical that aortic regions of differing permeability should have differing endothelial surface morphological characteristics. Thus, through use of SEM, the present study is concerned with examining aortic endothelial surface morphological characteristics in areas of differing permeability, i.e., blue and nonblue aortic segments, in an effort to determine whether aortic endothelial surface morphological differences do exist between these segments and whether such differences may be related to regional permeability characteristic of the aorta. MATERIALS
AND METHODS
Six male, 300-350-gm Wistar rats were employed in this study. Three hours prior to sacrifice 1 ml of 0.1% Evans blue (CI 23860, Baker Chemical Co.) in phosphate-buffered saline (PBS, pH 7.4) was injected via the tail vein. This solution had previously been sterilized by filtration through a Millipore filter (0.22 q). Each animal was sacrificed by cervical dislocation. After opening the thorax via a midline incision, the aorta, delimited by the heart and diaphragm, was carefully excised, opened longitudinally, and gently washed with additional PBS (pH 7.4) to remove adhering blood. The aorta was now secured by alpha cyanoacrylate glue to a glass microslide. Aortic regions showing differential Evans blue uptake, i.e., blue and white areas, were noted, mapped on an aortic diagram, and the total blueing area recorded. Based on differences in regional Evans blue uptake, aortas were divided into four segments: aortic arch, upper, middle, and lower thirds of the descending thoracic aorta. A 50-100 A gold coating was then vacuum evaporated onto the endothelial surface of each segment. Total elapsed time from sacrifice of animal to gold coating never exceeded 15 min. These segments were viewed at 25 kV using a JSM 50 A scanning electron microscope. RESULTS Aortic Evans Blue Uptake The overall pattern of aortic Evans blue uptake is similar to that reported in other species (Packham et al., 1967; Somer and Schwartz, 1971), with the greatest dye uptake occurring within the aortic arch and aortic ostea of the brachio-cephalic and left subclavian arteries. Essentially 85% of the total aortic arch lumenal surface area showed dye uptake, with streaking lines occurring along lines of blood flow. In the upper third of the thoracic aorta, approximately
RAT AORTIC
ENDOTHELIAL
SURF’ACE
MORPHOLOGY
25
40% of the lumenal surface area showed bhreing; no dye uptake was observed in either the middle or lower third of the thoracic aorta.
AOI%C arch. Figure I shows a representative SEM photomicrograph of the normal aortic arch surface topography. Endothelial folds and cross bridges are easily distinguishable. These folds, oriented in a oblique, longitudinal axis, have an average width of 13 ,.m, a fold to fold spacing of 20 mI and a depth of approximately 10 q. The cross bridges connecting two adjacent endo~elial folds average 22 w in length and 2-3 m in width and repeat at essentially 30-p intervals. The cross bridge is continuous with the endothelial surfaces of two endothelial folds. Interestingly, small projections ranging from 0.3 to 1 e in height are’ observable. At the encase of the left subclavian artery, the endo~eIia1 surface (Fig. 2) appears fess org~ized than at the body of the arch. Distinct endothdial foIds with cross bridges are present, however, with an increased number of endothehaf projections. Upper thoTac~c aorta. Figure 3 shows a similar representative SEM photomicrograph of the upper third of the descending thoracic aorta. Again distinct endothelial folds and cross bridges are present. The folds continue to run along the longitudinal axis of the aorta but appear to be less tortuous than those in the arch segment. The endotbelial folds and intercellular bridge size, however, appear to be decreasing with respect to the arch area. The average fold to fold spacing has decreased from 20 pm in the arch area to 10 pm in this segment. The number of cross bridges in this segment also appears to be much smaller than that observed in the arch area, The endothelid surface has a granular appearance not observed in the arch area. No endothelial projections are observabie. A general observation would be that the endothelium here is more simply organized than in the arch. Middle thoracic aorta, In the middle third of the descending thoracic aorta (Fig. 4) the endothelial foldings, while present, are less pronounced and less distinct than those of the arch and upper aortic segments, The folds continue to run along the longi~d~al axis of the aorta and have an approbate width of 5-IO p. The spacing between folds varies greatly in this area. No observable endotheliaf inter~elInlar bridges or endotheliat projections are noticeable in this area. Lower ~~~ac~c ~~$ta, In the lower third of the thoracic aorta (Fig. 5) the endothelial folds are still observable but are greatly reduced when compared to the above segments. As in preceding segments the endothelial folds continue to run along the longitudinal axis of the aorta. Many minor foldings of the endo~e~ial surface are present and in some cases give the appearance of being connected to adjacent endothelial folds. However, no distinct ~terendothe~~ cross bridges or endothelial projections are observable in this area. DISCUSSION The SEM observations presented in the current study on rat aorta are similar to and support previous descriptions of endo~elial folds and cross bridges made
26
KATORA
AND
HOLLIS
FIG. 1. SEM photomicrograph of rat ascending aorta and amtic arch (50-100 ing, x5000). Prominent endothelial folds (f) and cross bridges (c) are present. surface projections (p) are evident on both folds and cross bridges.
.& gold coatEndothelial
RAT AORTIC
ENDOTHELIAL
SURFACE
MORPNOLOGY
27
FIG, 2. SEM photomicro~aph of rat aortic arch at the left sub&&an ostea (5~100 PL gold coating, ~5000). Endothelial folds (f) and cross bridges (c) are present but are reduced in size. Note the large number of projections ( p ) on the end&he&al surface.
28
KATORA
AND HOLLIS
FIG. 3. SEM photomicrograph of normal rat aorta (the endothelial surface of the upper third of the descending thoracic aorta) (SO-100 il. gold coating, X5000). Characteristic endothelial folds (f) and cross bridges (c) are evident. In this region the endothelial surface appears grannular ( g ) .
RAT AORTIC
ENDOTHELIAL
SURFACE
MORPHOLOGY
2!)
FIG. 4. SEM photomicrograph of the endothelial surface of the middle third of the descending thoracic aorta (50-100 p\. gold coating, x5000). Endothelial folds (f) are present in this segment but are reduced both in size and regularity when compared to those in the upper segments of the aorta. No cross bridges are present.
30
KATORA
AND
HOLLIS
FIG. 5. SEM photomicrograph of descending thoracic aorta (50-100 ii gold coating, x5000). Endothelial folds are present in this segment but are smaller than those observed in the arch area and upper aortic segments. Many minifolds (m) that resemble cross bridges are present; however, no distinct cross bridges are apparent in this segment. A leukocyte (1) is seen penetrating the endothelium in the lower portion of this photomicrograph.
RAT AORTIC
ENDOTHELIAL
SURFACE
MORPHOLOGY
31
on rabbit (Shimamoto et al., 1969a,b; Sunaga et al., 1969a,b; Groniowski et al., 1971; Chisolm et al., 1973) and rat (Katora et al., 1974; Mauer et al., 1974) aortic endothelium. Considerable controversy, however, surrounds both the actual existence and significance of endothelial folds and cross bridges. The SEM and transmission electron (TEM) photomicrographs of Shimamoto et al. (1969a,b) indicate that these folds are composed of endothelial cells aligned in the long axis of the aorta and overlie the elastic lamellae. Additionally, they present photomicrographs that strongly suggest that intercellular cross bridges are, at least in some cases, actual protoplasmic connections between separate cells and speculate that these bridges represent an important regulatory component for passage of serous substances through interendothelial cell junctions into the vessel wall. However, Garbarsch and Christensen (1970) have presented SEM photomicrographs of rabbit aortas showing that the fold lines correspond to normal interendothelial silver-staining lines and failed to observe the presence of any cross bridges. Subsequently, they rejected the concept of either folds or cross bridges, suggesting these structures represent either fixation or dehydration artifacts (Christensen and Garbarsch, 1972). Similarly, Still and Dennison (1974) have failed to observe cross bridges in an SEM-TEM study dealing with aortic morphology in hypertensive rats. There is little question that fixation and dehydration procedures employed will profoundly influence topographical structure. While one can thus quite reasonably argue the relative merits of one fixation technique over another, we feel the surface morphological characteristics described in the present investigation are reconcilable with both proponents and opponents of folds and cross bridges, in as much as this study clearly establishes the fact that regional variations in surface morphology are present along the length of the aorta. Thus, while numerous folds and cross bridges are present in the ascending aorta and upper regions, the lower thoracic aorta shows greatly reduced folding and lacks cross bridges. Interestingly, the SEM observations of Christensen and Garbarsch (Christensen and Garbarsch, 1970; Garbarsch and Christensen, 1972) were made on midthoracic aortic segments; we note that folds in this region are less pronounced than those of the upper aortic region and that this region lacks cross bridges. Still and Dennison (1974) performed their observations on segments obtained from unspecified regions of rat aortas, and our SEM photomicrographs of lower thoracic aorta resemble those that they present. The SEM observations of Shimamoto et al. (1969a,b) and Sunaga et al. (1969a,b) show distinct folds and cross bridges that are almost identical to those that we observe in the arch and upper thoracic aorta. While most of the controversy over the existance of cross bridges has been limited to observations made on the rabbit aorta, it is reasonable to assume that a similar aortic endothelial surface topography exists in the rat. We thus conclude that some regions of the aorta contain cross bridges while others do not and, therefore, whether or not one observes cross bridges in aortic endothelium is dependent upon the aortic region observed. Regional variations in aortic permeability to various blood-borne macromolecules have been demonstrated in the pig (Packham et al., 1967; Somer and Schwartz, 1971; Bell et al., 1974a,b; Caplan et al., 1974), in the dog (McGill et al., 1957; Duncan et al., 1959), and most recently in the rat (Giacomelli and
32
KATORA
AND
HOLLIS
Wiener, 1974; Katora and Hollis, 1975). The blueing pattern observed in the present study is consistent with that previously reported in the above animal species; it is believed that focal blueing is the result of local endothelial injury induced by interactions of the vessel wall with heI~odynami~ stresses (Fry, 1969a,b), platelets, and leukocytes (Packham et al., 1967; J~r~ense~l et al., 1972). Recently Caplan et at. (1974) expiored the possibility that these differences in regional permeability caused by hemodynamic stresses might be associated with regional differences in endothelial silver staining as revealed by examination of en face preparations. They noted that endothelial cells in regions of blueing have essentially a three-fold greater frequency of line gaps than cells in regions of nonblueing and that the cell lines in these blue areas are thicker. They suggest that the thicker lines might reflect wider intercellular junctio~~s and that the gaps may represent either defects in cell junctions or cytoplasmic projections overlying complex junctions that bIock silver nitrate penetration. The SEM observations of the present investigation tend to support the latter possibility for the following reasons: The width of the gaps is in many cases similar to the width of the cross bridges; cross bridges are most numerous in the upper aortic regions; each cross bridge connecting adjacent folds would by necessity pass over one if not more intercellular junctions, thereby mechanically preventing silver nitrate penetration. As previously mentioned, Shimamoto and co-workers (1969a,b) have proposed that cross bridges represent an important structural component in the endothelial regulation of transmural permeability. They visualize these bridges as maintaining intercellular junctions within a certain functional pore size during vessel distention and recoil and have shown that cross bridge “contraction” occurs following a single iv cholesterol challenge. While hard evidence supporting this concept is notably lacking, it is interesting that regions of the aorta having the greatest number of cross bridges are aIso the preferential regions for development of atheromata. The observations presented in this current study indicate that cross bridges are present in regions of highest aortic transmural permeability. The significance of these bridges with respect to either regulation of vessel wall permeability or regional variations in permeability, however, must await further examination, Another interesting observation in the present study was that of the endothelial surface projections. Smith et aE. (1971) have described these projections on the endothelial surface of dog pulmonary artery. In their study these projections varied from 0.3 pm to 3.0 pn in length. More recently, Still and Dennison (1974) have shown the presence of these projections in rat aortic endothelium. In the present study these projections were noted only in aortic regions corresponding to those of enhanced Evans blue uptake, notably lacking in the middle and lower thirds of the thoracic aorta. It should be noted, however, that the physiological signi~c~ce of these projections is unknown to date. In summary, this investigation has established that regional variations in aortic surface morphology are present within the rat aorta. In those aortic regions having locally elevated permeability to serous substances, distinct, prominent endothelial folds and numerous cross bridges are present, while in regions of reduced permeability these folds and cross bridges are either reduced or absent.
RAT AORTIC
ENDOT~LIAL
SURFA~
~~ORPI~O~GY
33
The morphological features described appear reconcilable in light of the current controversy existing with respect to endothelial folds and cross bridges. ACKNOWLEDGMENT The authors gratefully
acknowledge
the technical assistance of Charles Maurer.
REFERENCES F. P., ADAMSON, I. L., and SCHWARTZ, C. J. (1974a). Aortic endothelial permeability to albumin: Focal and regional patterns of uptake and transmural distribution of 1311albumin in the young pig. Exp. Mol. Pathol. 20, 57-68. BELL, F. P., GALLUS, A., and SCHWARTZ, C. J. (1947b). Aortic endothelial permeability to fibrinogen: Focal and regional patterns of uptake and transmural distribution of “*Ifibrinogen in the young pig. Exp. Mol. Pathol. 20, 281-292. CAPLAN, B. A., GERRITY, R. G., and SCHWARTZ, C. J. (1974). Endothelial ceil morphoIogy in focal areas of in wivo Evans blue uptake in the young pig aorta. I. Quantitative light microscopic findings. Exp. Mol. Path&. 21, 102-117. CAPLAN, B. A., and SCHWARTZ, C. J. (1973). Increased endothelial cell turnover in areas of in viuo Evans blue uptake in the pig aorta. Atherosclerosis 17, 401417. CHISOLM, G. M., GAINER, J. L., and STONER, G. E. ( 1973). SEM (scanning electron microscope) studies of aortic structure. Angiologica 10, 10-14. CHRISTENSEN, B. C., and GARBARSCH, C. (1972). A scanning electron microscopic (SEM) study on the endothelium of the normal rabbit aorta. AngioEogira 9, 15-26. DUNCAN, L. E., and BUCK, K. ( 1961). Passage of labeled albumin into canine aortic wall in viva and in u&o. Amer. J. Physiol. 200, 622-624. DUNCAN, L. E., CORNFIELD, J-, and BUCK, K. (1959). Circulation of labeled albumin through the aortic wall of the dog. C&c. Res. 7, 390-397. DUNCAN, L. E., CORNFIELD, J., and BUCK, K. (1962). The effect of blood pressure on &e passage of labeled plasma albumin into canine aortic wall. J. Clin. Inoest. 41, 1537-1545. FRIEDMAN, M., and BYERS, S. 0. ( 1963). Endothelial permeability in atherosclerosis. Arch. Pathol. 76, 99-105. FRY, D. L. (1969a). Certain chemorheologic considerations regarding the blood vascular interface with particular reference to coronary artery disease. Circulation 40, 3857. FRY, D. L. (1969b). Certain histological and chemical responses of the vascular interface to acutely induced mechanical stress in the aorta of the dog. Circ. Res. 24, 93-108. FRY, D. L. (1973). Responses of the arterial wall to certain physical factors. In “Atherogenesis: Initiating Factors,” Ciba Foundation S~posi~ 12, pp. 93-120. American Elsevier, New York. GARBARSCH, C., and CHRISTENSEN, B. C. (1970). Scanning electron microscopy of sortie endothelial cell boundaries after staining with silver nitrate. Angiologica 7, 365-373. GIACOMELLI, F., and WIENER, J. ( 1974). R e g’ronal variation in the permeability of rat thoracic aorta. Amer. J. Pathol. 75, 513-528. GRONIOWSKI, J,, BICZYSKOWA, W., and WALSKI, M. (1971). Scanning electron microscopic observations on the surface of vascular endothelium. Folia Histochem. Cytochem. 9, 243-246. JPRGENSEN, L,, PACKHAM, M. A., ROWSELL, H. C., and MUSTARD, J. F. (1972). Deposition of formed elements of blood on the intima and signs of intimal injury in the aorta of rabbit, pig and man. Lab. fnuest. 27, 341350. KATORA, M. E., and HOLLIS, T. M. ( 1975). A simple fluorescent method for quantitative determination of aortic protein uptake. J. Appl. Physiol. 39, 145-149. KATORA, M. El., MAWR, C., and HOLLIS, T. M. ( 1974). Effect of short term hypertension on rat aortic endothelial surface topography. PTOC. Elect. Microsc. Sot. Amer. 32, 150-151. KLYNSTRA, F. B., and B&THE,, C. J. F. (I970). P ermeability patterns in pig aorta. Atherosclerosis 11, 451-462.
BELL,
MATJEZR, C., KATORA, M. E., and HOLLIS, T. XI. (1974). Effect of ventromedial laparotomy on aortic arch endothehal surface topography. Proc. Elect. Micros. Sot. 32, 32-33. MCGKL, H. C., GEER, J. C., and HOLMAN, R. L. (1957). Sites of vascalar V~~Ineral~~lity in dogs demonstrated by Evans Blue. ArcIs. Fat~zol. 64, 303-311. PACKHAM, M. A., RO~SELL, H. C., J#RGENSEN, L., and MUSTARD, J. F. (1967). Loeahzed protein accumulation in the wall of the aorta. Exp. MOE. Pathol. 7, 214-232. SHIMAMOTO, T., YAMASHITA, Y., and SUNAGA, T. (1969a). Scanning electron microscopic observation of endothelial surface of heart and bIood vessels. Proc. Jay. AcarZ. 45, 507-511. SHIMAMOTO, T., YAMASHITA, Y., NUAIANO, F., and SUNAGA, T. (1969b). The endothelial cell damages of pre-atheromatous and atheromatous lesions observed by scanning electron microscope. Proc. Jap. Acad. 45, ‘761-766. S~~ITH, U., RYAN, J. W., MICHIE, A. A., and SMITH, D. S. ( 1971). Endothelial projections as revealed by scanning electron microscope. Science 173, 925-927. SOMER, J. B., and SCHWARTZ, C. J. (1971). Focal 3H-cholesterol uptake in the pig aorta. AtheroscleTos~ 13, 293304. SOMER, J. B., and SCHWARTZ, C. J. ( 1972). Focal ‘H-cholesterol uptake in the pig aorta. Part 2. Dist~bution of ‘H-cholesterol across the aortic wall in areas of high and low uptake in vivo. Athe~o~c~ero~s 16, 377388. STILL, W. J. S., and DENNISON, S. (1974). The arterial endothelium of the hypertensive rat. Arch. Pathol. 92, 337-342. SUNAGA, T., YAXfAsrrrT.4, Y., and SHIMAMOTO, T. (1969a). The intercellular bridge of vascular endothelium. Proc. Jap. Acad. 45, 627-631. SUNAGA, T., YAMASHITA, Y., and SHIMAMATO, T. ( 196913). Epinephrine effect on arterial endothelial cells observed by scanning electron microscope. Proc. Jap. Acad. 45, 808-813.
AND
Regional
XfOLECULAR
PATHOLOGY
24, 23-34
( 19%)
Variation in Rat Aortic Endothelial Surface Relationship to Regional Aortic Permeability ~IC~~~.KATO~A~T~ODO~
Biology
~epe~~~,
Received
208 Life University April
M.HOLL~S
Sceinces I, The Pen~~~l~~~~i~ Pu&, Penns~~vaniu 16802
28, 1975,
morphology:
and
in revised
fom
June
State
Uniuersit~,
9, 1975
The regional variation of rat aortic lumenal surface topography has been examined by employing techniques of scanning electron microscopy. This study was undertaken in an attempt to gain additional insight into the current controversy regarding endothelial folds and cross bridges and their possible role with respect to regulation of vessel transmural permeability. Within the ascending aorta, aortic arch, and upper third of the thoracic aorta distinct, prominent endothelial folds connected by interlinking cross bridges were easily identifiable. The middle third of the thoracic aorta contained less prominent endothelial folds; photomicrographs from these regions consistently failed to reveal the presence of any interfold cross bridges. In the lower third of the thoracic aorta, endothelial folds were notably lacking. These differences in regional aortic surface topography have been discussed in light of known regional differences in aortic wall permeability and with respect to regional differences in appearance of endothelial silver-staining lines.
INTRODUCTION Through use of vital dyes, fluorescent markers, and radioisotopes it is now generally recognized that regional variations in aortic pe~eabili~ exist within most mammalian species (Duncan et al., 1959, 196I, 1962; J$rgensen et al., 1970; Klynstra and Biittcher, 1970; Katora and Hollis, 1975). Regions of increased permeability include such areas as the inner curvatnre of the aortic arch, sinuses of Valsalva, orifices of vessels to the head and neck, and thoracic aortic intercostal ostea (Friedman and Byers, 1963; Packham et at., 1967; Klynstra and Bottcher, 1970; Jpirgensenet al., 1972; Giacomelli and Wiener, 1974). In numerous studies, among which are those of Fry ( 1969a,b, 1973), these areas have been identified by increased uptake of Evans blue dye and coincide with regions in which hemodynamic stressesare locally elevated. It has been proposed that these regions represent areas in which hemodynamic-mediated endothelial injury has occurred. Aortic regions showing focal blueing are associated with increased uptake of fibrinogen (Bell et al., 1974b), albumin (Packham et al., 1967; Bell et al., 1974a), aud unesterified cholesterol (Somer and Schwartz, 1972); such regions also show increased endothelial turnover ( Caplan and Schwartz, 1973). Recently, Caplan et al. (1974) h ave described differences in thickness and the number of gaps * Supported
by
NSF
Grant
GR-38136.
Send
reprint 23
Copyright All tights
1976 by Academic Press, Inc. reproduction in any form reserved.
requests
to Dr.
Theodore
M.
Hollis.
KATORA
24
AND HOLLIS
located within silver staining lines of Haiitchen preparations of endothelium obtained from areas of aortic blueing and nonblueing in an attempt to determine whether morphological differences in these lines (intercellular junctions) can account for this altered aortic transmural permeability. In a series of scanning electron microscopy (SEM) studies, Shimamoto and co-workers (1969a) have suggested that endothelial intercellular cross bridges are vital and active components in the regulation of aortic endothelial permeability. Since the endothelium is now recognized as occupying a central role in regulation of aortic transmural permeability, and in light of these previous studies (Shimamoto et al., 1969a,b; Giacomelli and Wiener, 1974; Caplan et al., 1974), it would seem logical that aortic regions of differing permeability should have differing endothelial surface morphological characteristics. Thus, through use of SEM, the present study is concerned with examining aortic endothelial surface morphological characteristics in areas of differing permeability, i.e., blue and nonblue aortic segments, in an effort to determine whether aortic endothelial surface morphological differences do exist between these segments and whether such differences may be related to regional permeability characteristic of the aorta. MATERIALS
AND METHODS
Six male, 300-350-gm Wistar rats were employed in this study. Three hours prior to sacrifice 1 ml of 0.1% Evans blue (CI 23860, Baker Chemical Co.) in phosphate-buffered saline (PBS, pH 7.4) was injected via the tail vein. This solution had previously been sterilized by filtration through a Millipore filter (0.22 q). Each animal was sacrificed by cervical dislocation. After opening the thorax via a midline incision, the aorta, delimited by the heart and diaphragm, was carefully excised, opened longitudinally, and gently washed with additional PBS (pH 7.4) to remove adhering blood. The aorta was now secured by alpha cyanoacrylate glue to a glass microslide. Aortic regions showing differential Evans blue uptake, i.e., blue and white areas, were noted, mapped on an aortic diagram, and the total blueing area recorded. Based on differences in regional Evans blue uptake, aortas were divided into four segments: aortic arch, upper, middle, and lower thirds of the descending thoracic aorta. A 50-100 A gold coating was then vacuum evaporated onto the endothelial surface of each segment. Total elapsed time from sacrifice of animal to gold coating never exceeded 15 min. These segments were viewed at 25 kV using a JSM 50 A scanning electron microscope. RESULTS Aortic Evans Blue Uptake The overall pattern of aortic Evans blue uptake is similar to that reported in other species (Packham et al., 1967; Somer and Schwartz, 1971), with the greatest dye uptake occurring within the aortic arch and aortic ostea of the brachio-cephalic and left subclavian arteries. Essentially 85% of the total aortic arch lumenal surface area showed dye uptake, with streaking lines occurring along lines of blood flow. In the upper third of the thoracic aorta, approximately
RAT AORTIC
ENDOTHELIAL
SURF’ACE
MORPHOLOGY
25
40% of the lumenal surface area showed bhreing; no dye uptake was observed in either the middle or lower third of the thoracic aorta.
AOI%C arch. Figure I shows a representative SEM photomicrograph of the normal aortic arch surface topography. Endothelial folds and cross bridges are easily distinguishable. These folds, oriented in a oblique, longitudinal axis, have an average width of 13 ,.m, a fold to fold spacing of 20 mI and a depth of approximately 10 q. The cross bridges connecting two adjacent endo~elial folds average 22 w in length and 2-3 m in width and repeat at essentially 30-p intervals. The cross bridge is continuous with the endothelial surfaces of two endothelial folds. Interestingly, small projections ranging from 0.3 to 1 e in height are’ observable. At the encase of the left subclavian artery, the endo~eIia1 surface (Fig. 2) appears fess org~ized than at the body of the arch. Distinct endothdial foIds with cross bridges are present, however, with an increased number of endothehaf projections. Upper thoTac~c aorta. Figure 3 shows a similar representative SEM photomicrograph of the upper third of the descending thoracic aorta. Again distinct endothelial folds and cross bridges are present. The folds continue to run along the longitudinal axis of the aorta but appear to be less tortuous than those in the arch segment. The endotbelial folds and intercellular bridge size, however, appear to be decreasing with respect to the arch area. The average fold to fold spacing has decreased from 20 pm in the arch area to 10 pm in this segment. The number of cross bridges in this segment also appears to be much smaller than that observed in the arch area, The endothelid surface has a granular appearance not observed in the arch area. No endothelial projections are observabie. A general observation would be that the endothelium here is more simply organized than in the arch. Middle thoracic aorta, In the middle third of the descending thoracic aorta (Fig. 4) the endothelial foldings, while present, are less pronounced and less distinct than those of the arch and upper aortic segments, The folds continue to run along the longi~d~al axis of the aorta and have an approbate width of 5-IO p. The spacing between folds varies greatly in this area. No observable endotheliaf inter~elInlar bridges or endotheliat projections are noticeable in this area. Lower ~~~ac~c ~~$ta, In the lower third of the thoracic aorta (Fig. 5) the endothelial folds are still observable but are greatly reduced when compared to the above segments. As in preceding segments the endothelial folds continue to run along the longitudinal axis of the aorta. Many minor foldings of the endo~e~ial surface are present and in some cases give the appearance of being connected to adjacent endothelial folds. However, no distinct ~terendothe~~ cross bridges or endothelial projections are observable in this area. DISCUSSION The SEM observations presented in the current study on rat aorta are similar to and support previous descriptions of endo~elial folds and cross bridges made
26
KATORA
AND
HOLLIS
FIG. 1. SEM photomicrograph of rat ascending aorta and amtic arch (50-100 ing, x5000). Prominent endothelial folds (f) and cross bridges (c) are present. surface projections (p) are evident on both folds and cross bridges.
.& gold coatEndothelial
RAT AORTIC
ENDOTHELIAL
SURFACE
MORPNOLOGY
27
FIG, 2. SEM photomicro~aph of rat aortic arch at the left sub&&an ostea (5~100 PL gold coating, ~5000). Endothelial folds (f) and cross bridges (c) are present but are reduced in size. Note the large number of projections ( p ) on the end&he&al surface.
28
KATORA
AND HOLLIS
FIG. 3. SEM photomicrograph of normal rat aorta (the endothelial surface of the upper third of the descending thoracic aorta) (SO-100 il. gold coating, X5000). Characteristic endothelial folds (f) and cross bridges (c) are evident. In this region the endothelial surface appears grannular ( g ) .
RAT AORTIC
ENDOTHELIAL
SURFACE
MORPHOLOGY
2!)
FIG. 4. SEM photomicrograph of the endothelial surface of the middle third of the descending thoracic aorta (50-100 p\. gold coating, x5000). Endothelial folds (f) are present in this segment but are reduced both in size and regularity when compared to those in the upper segments of the aorta. No cross bridges are present.
30
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FIG. 5. SEM photomicrograph of descending thoracic aorta (50-100 ii gold coating, x5000). Endothelial folds are present in this segment but are smaller than those observed in the arch area and upper aortic segments. Many minifolds (m) that resemble cross bridges are present; however, no distinct cross bridges are apparent in this segment. A leukocyte (1) is seen penetrating the endothelium in the lower portion of this photomicrograph.
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ENDOTHELIAL
SURFACE
MORPHOLOGY
31
on rabbit (Shimamoto et al., 1969a,b; Sunaga et al., 1969a,b; Groniowski et al., 1971; Chisolm et al., 1973) and rat (Katora et al., 1974; Mauer et al., 1974) aortic endothelium. Considerable controversy, however, surrounds both the actual existence and significance of endothelial folds and cross bridges. The SEM and transmission electron (TEM) photomicrographs of Shimamoto et al. (1969a,b) indicate that these folds are composed of endothelial cells aligned in the long axis of the aorta and overlie the elastic lamellae. Additionally, they present photomicrographs that strongly suggest that intercellular cross bridges are, at least in some cases, actual protoplasmic connections between separate cells and speculate that these bridges represent an important regulatory component for passage of serous substances through interendothelial cell junctions into the vessel wall. However, Garbarsch and Christensen (1970) have presented SEM photomicrographs of rabbit aortas showing that the fold lines correspond to normal interendothelial silver-staining lines and failed to observe the presence of any cross bridges. Subsequently, they rejected the concept of either folds or cross bridges, suggesting these structures represent either fixation or dehydration artifacts (Christensen and Garbarsch, 1972). Similarly, Still and Dennison (1974) have failed to observe cross bridges in an SEM-TEM study dealing with aortic morphology in hypertensive rats. There is little question that fixation and dehydration procedures employed will profoundly influence topographical structure. While one can thus quite reasonably argue the relative merits of one fixation technique over another, we feel the surface morphological characteristics described in the present investigation are reconcilable with both proponents and opponents of folds and cross bridges, in as much as this study clearly establishes the fact that regional variations in surface morphology are present along the length of the aorta. Thus, while numerous folds and cross bridges are present in the ascending aorta and upper regions, the lower thoracic aorta shows greatly reduced folding and lacks cross bridges. Interestingly, the SEM observations of Christensen and Garbarsch (Christensen and Garbarsch, 1970; Garbarsch and Christensen, 1972) were made on midthoracic aortic segments; we note that folds in this region are less pronounced than those of the upper aortic region and that this region lacks cross bridges. Still and Dennison (1974) performed their observations on segments obtained from unspecified regions of rat aortas, and our SEM photomicrographs of lower thoracic aorta resemble those that they present. The SEM observations of Shimamoto et al. (1969a,b) and Sunaga et al. (1969a,b) show distinct folds and cross bridges that are almost identical to those that we observe in the arch and upper thoracic aorta. While most of the controversy over the existance of cross bridges has been limited to observations made on the rabbit aorta, it is reasonable to assume that a similar aortic endothelial surface topography exists in the rat. We thus conclude that some regions of the aorta contain cross bridges while others do not and, therefore, whether or not one observes cross bridges in aortic endothelium is dependent upon the aortic region observed. Regional variations in aortic permeability to various blood-borne macromolecules have been demonstrated in the pig (Packham et al., 1967; Somer and Schwartz, 1971; Bell et al., 1974a,b; Caplan et al., 1974), in the dog (McGill et al., 1957; Duncan et al., 1959), and most recently in the rat (Giacomelli and
32
KATORA
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Wiener, 1974; Katora and Hollis, 1975). The blueing pattern observed in the present study is consistent with that previously reported in the above animal species; it is believed that focal blueing is the result of local endothelial injury induced by interactions of the vessel wall with heI~odynami~ stresses (Fry, 1969a,b), platelets, and leukocytes (Packham et al., 1967; J~r~ense~l et al., 1972). Recently Caplan et at. (1974) expiored the possibility that these differences in regional permeability caused by hemodynamic stresses might be associated with regional differences in endothelial silver staining as revealed by examination of en face preparations. They noted that endothelial cells in regions of blueing have essentially a three-fold greater frequency of line gaps than cells in regions of nonblueing and that the cell lines in these blue areas are thicker. They suggest that the thicker lines might reflect wider intercellular junctio~~s and that the gaps may represent either defects in cell junctions or cytoplasmic projections overlying complex junctions that bIock silver nitrate penetration. The SEM observations of the present investigation tend to support the latter possibility for the following reasons: The width of the gaps is in many cases similar to the width of the cross bridges; cross bridges are most numerous in the upper aortic regions; each cross bridge connecting adjacent folds would by necessity pass over one if not more intercellular junctions, thereby mechanically preventing silver nitrate penetration. As previously mentioned, Shimamoto and co-workers (1969a,b) have proposed that cross bridges represent an important structural component in the endothelial regulation of transmural permeability. They visualize these bridges as maintaining intercellular junctions within a certain functional pore size during vessel distention and recoil and have shown that cross bridge “contraction” occurs following a single iv cholesterol challenge. While hard evidence supporting this concept is notably lacking, it is interesting that regions of the aorta having the greatest number of cross bridges are aIso the preferential regions for development of atheromata. The observations presented in this current study indicate that cross bridges are present in regions of highest aortic transmural permeability. The significance of these bridges with respect to either regulation of vessel wall permeability or regional variations in permeability, however, must await further examination, Another interesting observation in the present study was that of the endothelial surface projections. Smith et aE. (1971) have described these projections on the endothelial surface of dog pulmonary artery. In their study these projections varied from 0.3 pm to 3.0 pn in length. More recently, Still and Dennison (1974) have shown the presence of these projections in rat aortic endothelium. In the present study these projections were noted only in aortic regions corresponding to those of enhanced Evans blue uptake, notably lacking in the middle and lower thirds of the thoracic aorta. It should be noted, however, that the physiological signi~c~ce of these projections is unknown to date. In summary, this investigation has established that regional variations in aortic surface morphology are present within the rat aorta. In those aortic regions having locally elevated permeability to serous substances, distinct, prominent endothelial folds and numerous cross bridges are present, while in regions of reduced permeability these folds and cross bridges are either reduced or absent.
RAT AORTIC
ENDOT~LIAL
SURFA~
~~ORPI~O~GY
33
The morphological features described appear reconcilable in light of the current controversy existing with respect to endothelial folds and cross bridges. ACKNOWLEDGMENT The authors gratefully
acknowledge
the technical assistance of Charles Maurer.
REFERENCES F. P., ADAMSON, I. L., and SCHWARTZ, C. J. (1974a). Aortic endothelial permeability to albumin: Focal and regional patterns of uptake and transmural distribution of 1311albumin in the young pig. Exp. Mol. Pathol. 20, 57-68. BELL, F. P., GALLUS, A., and SCHWARTZ, C. J. (1947b). Aortic endothelial permeability to fibrinogen: Focal and regional patterns of uptake and transmural distribution of “*Ifibrinogen in the young pig. Exp. Mol. Pathol. 20, 281-292. CAPLAN, B. A., GERRITY, R. G., and SCHWARTZ, C. J. (1974). Endothelial ceil morphoIogy in focal areas of in wivo Evans blue uptake in the young pig aorta. I. Quantitative light microscopic findings. Exp. Mol. Path&. 21, 102-117. CAPLAN, B. A., and SCHWARTZ, C. J. (1973). Increased endothelial cell turnover in areas of in viuo Evans blue uptake in the pig aorta. Atherosclerosis 17, 401417. CHISOLM, G. M., GAINER, J. L., and STONER, G. E. ( 1973). SEM (scanning electron microscope) studies of aortic structure. Angiologica 10, 10-14. CHRISTENSEN, B. C., and GARBARSCH, C. (1972). A scanning electron microscopic (SEM) study on the endothelium of the normal rabbit aorta. AngioEogira 9, 15-26. DUNCAN, L. E., and BUCK, K. ( 1961). Passage of labeled albumin into canine aortic wall in viva and in u&o. Amer. J. Physiol. 200, 622-624. DUNCAN, L. E., CORNFIELD, J-, and BUCK, K. (1959). Circulation of labeled albumin through the aortic wall of the dog. C&c. Res. 7, 390-397. DUNCAN, L. E., CORNFIELD, J., and BUCK, K. (1962). The effect of blood pressure on &e passage of labeled plasma albumin into canine aortic wall. J. Clin. Inoest. 41, 1537-1545. FRIEDMAN, M., and BYERS, S. 0. ( 1963). Endothelial permeability in atherosclerosis. Arch. Pathol. 76, 99-105. FRY, D. L. (1969a). Certain chemorheologic considerations regarding the blood vascular interface with particular reference to coronary artery disease. Circulation 40, 3857. FRY, D. L. (1969b). Certain histological and chemical responses of the vascular interface to acutely induced mechanical stress in the aorta of the dog. Circ. Res. 24, 93-108. FRY, D. L. (1973). Responses of the arterial wall to certain physical factors. In “Atherogenesis: Initiating Factors,” Ciba Foundation S~posi~ 12, pp. 93-120. American Elsevier, New York. GARBARSCH, C., and CHRISTENSEN, B. C. (1970). Scanning electron microscopy of sortie endothelial cell boundaries after staining with silver nitrate. Angiologica 7, 365-373. GIACOMELLI, F., and WIENER, J. ( 1974). R e g’ronal variation in the permeability of rat thoracic aorta. Amer. J. Pathol. 75, 513-528. GRONIOWSKI, J,, BICZYSKOWA, W., and WALSKI, M. (1971). Scanning electron microscopic observations on the surface of vascular endothelium. Folia Histochem. Cytochem. 9, 243-246. JPRGENSEN, L,, PACKHAM, M. A., ROWSELL, H. C., and MUSTARD, J. F. (1972). Deposition of formed elements of blood on the intima and signs of intimal injury in the aorta of rabbit, pig and man. Lab. fnuest. 27, 341350. KATORA, M. E., and HOLLIS, T. M. ( 1975). A simple fluorescent method for quantitative determination of aortic protein uptake. J. Appl. Physiol. 39, 145-149. KATORA, M. El., MAWR, C., and HOLLIS, T. M. ( 1974). Effect of short term hypertension on rat aortic endothelial surface topography. PTOC. Elect. Microsc. Sot. Amer. 32, 150-151. KLYNSTRA, F. B., and B&THE,, C. J. F. (I970). P ermeability patterns in pig aorta. Atherosclerosis 11, 451-462.
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