209
Atherosclerosis,89 (1991) 209-221 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0021.9150/91/$03.50 ADONIS
002191509100152L
ATHERO
04682
Change in endothelial cell morphology at arterial branch sites caused by a reduction of intramural stress * Joseph
W. Baker
‘, Mano J. Thubrikar ‘, Jayashri S. Parekh and Stanton P. Nolan ’
‘, Michael
S. Forbes
2
’Department ofSurgery and 2 Department of Physiology, &ice&y
of Virginia Health Sciences Center, Charlottesl,ille, Virginia 22908 (U.S.A.)
(Received 18 March, 1991) (Revised, received 9 May, 1991) (Accepted 10 May, 1991)
Summary Arterial branch sites have very high intramural stresses at physiologic intraluminal pressures; the same sites have a predilection for atherosclerosis. The effect of intramural stress on endothelial cell morphology was investigated. Five rabbits had permanent casts placed around a segment of the abdominal
aorta-left renal artery branch area during controlled hypotension, thus reducing intramural stress without narrowing the lumen. These five animals, and three normal rabbits, were sacrificed after 4-8 weeks, and the vessels were perfused with buffered 2.5% glutaraldehyde for 2 h at 100 mm Hg pressure. The aortas were examined by scanning electron microscopy. In normal aortas, the distal region of the ostia of the left renal and celiac arteries just beyond the flow divider displayed many morphologically altered endothelial cells ranging from spindle shape to cobble-stone shape. The same aortic area of casted rabbits, as well as the straight abdominal aorta in all rabbits, showed a smooth surface of endothelial cells with intact cell borders and no morphologically altered cells. At branch sites, the occurrence of morphologically altered endothelial cells may be due to increased intramural stress. When intramural stress is reduced, the morphology of branch endothelial cells changes to resemble that of the unbranched regions. In conclusion, endothelial cell morphology changes in response to changes in intramural stress.
Key words:
Endothelial
morphology;
Arterial
branch;
Intramural
stress; Atherosclerosis
Introduction * This work was presented
in part
at the 1987 and
1989
FASEB meetings. Correspondence to: Mano J. Thubrikar, Ph.D. Department of Surgery, MR4 Box 3111 University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, U.S.A.
Endothelial
cells are known to respond to a
variety of mechanical stimuli. In cell culture studies, endothelial cells align in the direction of flow,
210 i.e., the cells become elongated and oriented with their long axis parallel to the direction of flow [l-4]. Endothelial cells cultured on a compliant membrane undergoing cyclic stress become oriented perpendicular to the direction of stretching [.5-71. Endothelial cells, therefore, orient not only in response to flow but also in response to deformation of the underlying substrate. In arteries, endothelial cells are aligned along the axis of the artery [S-10]. This orientation could result from the synergistic actions of blood flow and arterial stretch. In vivo the artery undergoes stretching in the circumferential direction, which would tend to orient the cells along the length of the artery. The direction of blood flow is along the length of the artery, which would also orient the cells in that direction. In the leaflets of the aortic valve.
the endothelial cells align not in the direction of blood flow, but perpendicular to it [ll]. In this case, it was suggested that the cells are oriented in the direction of the maximum tensile stress in the leaflet [ll]. Endothelial cell injury has been implicated in the process of atherogenesis. Since atherosclerotic lesions form at sites of arterial branching in both humans [12,13] and animals [14,15], an understanding of endothelial cell morphology at these sites would be of great value. It has previously been reported that endothelial cell morphology in branching regions is different from that in straight regions [16-211 and that this difference may be due to the complex blood flow patterns occurring in localized regions of the branches [20-221. The arterial branch area is
Fig. 1. Maximum principal stress contours on the inner surface of the artery. These stresses are due to an incremental pressure loading of 40 mm Hg. The stress increases from one contour to the next towards the ostium. The stress increase occurs at both the proximal and distal regions of the ostium. The numbers represent stress values in MPa. These results were obtained from a bovine circumflex coronary arterial branch [23]. (This figure was taken from our publication, J. Biomechanics, 23;1989:15.)
211 unique in other ways as well. Intramural stresses, which are produced by the intraluminal blood pressure, are quite unevenly distributed in the branch area. Intramural stresses are tensile stresses, and they are responsible for the stretching of the artery. They are produced by blood pressure, not by blood flow, and they occur throughout the thickness of the arterial wall. We have found that intramural stresses in branch regions are 3-7 times those in nonbranch (straight) regions [23-2.51. Intramural stress is also highest on the inner surface of arteries (Fig. 1) [23]. Intramural stress is high at branch sites because the ostium of the arterial branch creates a reduction in the quantity of arterial tissue of the main artery. High intramural stresses at the branch are accompanied by significantly increased stretching (high strains) of the wall locally [23]. These high stresses and high strains should be associated with altered endothelial cell morphology at the branch site. We have observed that when intramural stress is reduced in branch areas, there is an inhibition of the development of atherosclerotic lesions [26,27]. In the present study, we investigate whether there is a difference between endothelial cell morphology in regions of high as opposed to regions of low intramural stress and whether the morphology changes when intramural stress is reduced. Methods
Endothelial cell morphology was studied with scanning electron microscopy. Intramural stress
Fig. 2. (A) Diagram of a rabbit abdominal aorta and major branches with a cast around the aorta - left renal artery segment. (B) perpendicular forces are buttressed by the rigid cast, and intramural stress is reduced. (0 parallel forces related to blood flow are not significantly altered by the cast.
Celiac A. Superior Mesenteric
Rtght Renal A
A.
Lett Kenal
A
Blood Flou
B
Fig. 3. (A) Schematic presentation of the abdominal aorta showing four major arterial branches under study. Arterial branch areas and straight segments were examined by SEM. (B) Schematic presentation of the opened aorta showing the ostium of a branch. SEM photographs were taken at the distal lip of the ostium (69) and at a straight region of the aorta ( ? ? ).
in the arterial branch area was reduced by placement of a periarterial cast. Five adult New Zealand white rabbits (body wt. 4.0-4.5 kg) were anesthetized with intramuscular ketamine (50 mg/kg) and acepromazine (5 mg/kg), and maintained on l-2% halothane by face mask. Intravenous and intra-arterial catheters were placed percutaneously in ear vessels for intravenous infusion and arterial blood pressure measurement. A midline laparotomy was performed, and a segment of the abdominal aorta and proximal left renal artery was isolated. The aorta and left renal artery were dissected free of fatty tissue circumferentially and a small piece of metallic foil was placed posterior to the branch area to form the back of the cast. The aorta-left renal artery branch was then covered with warm saline for 10 min to resolve any vasospasm. The mean arterial blood pressure was then lowered to 40 mm Hg and maintained by the intravenous infusion of nitroprusside. During this state of controlled hypotension, liquid methyl-methacrylate dental acrylic compound was poured around the isolated segment of aorta and proximal left renal artery. The cast was allowed to harden while the blood pressure was maintained at a constant level, and the nitroprusside was then discontinued. This produced a circumferential cast which encased the segment of aorta and proximal left renal artery. The rabbits underwent standard closure of the laparotomy and were allowed to recover for 4-8 weeks on a standard rabbit diet. Three control (unoperated) adult New Zealand white rabbits
Fig. 4. (A-B) SEM photomicrographs smooth. Endothelial cells have uniform direction of blood flow. A and B are noted. Note: Figs. 4-6: magnification
of the luminal surface of a straight portion of the abdominal aorta. The intimal surface is size and they are flat and elongated. Cell borders are intact and the cells are oriented in the different rabbits. A slight variation in the appearance of normal endothelial cells may be is 1600x and the direction of blood flow is approximately from top to bottom of the photomicrographs.
4.7-5.1 kg) were also used to study endothelial morphology in the normal branch area. The technique described above has been used by us previously to reduce intramural stress [27]. Intramural stress is related directly to intraarterial pressure. In the cylindrical segment of the artery
(wt.
Pr Pr uC= - and u, = 2T T where a, and (+, represent stress in the circumferential and longitudinal direction and P, R and T represent arterial pressure, radius and thickness, respectively. In branch areas, stress distribu-
tion is complex (Fig. 1) and stress is increased by 300-700% mainly as a result of the presence of a branch ostium 123-251. When blood pressure is reduced, stress becomes lower and the diameter of the artery decreases slightly, probably less than 4%. When a cast is placed at a reduced blood pressure the artery remains in this configuration and consequently in this state of decreased stress. When blood pressure is allowed to rise, the artery is prevented from expanding because of the rigid cast and a low intramural stress is maintained in the casted segment of the artery. Our previous studies showed that cast placement did not cause measurable narrowing of the arterial lumen or a decrease of arterial wall thickness [27]. Because
213
Fig. 4. (contimied)
the cast is rigid, it does eliminate pulsatile expansion of the artery; however, it is not expected to alter the regions of high and low fluid shear (Fig. 2). Most of the studies of fluid shear have been performed using rigid glass models of arteries [28,29]. It is possible that the cast may produce slight changes in pressure waveform or pulse pressure; however, the influence of these changes must be minor, because, when casts are placed at high systemic pressures (9.5 mm Hg), there is no reduction of intramural stress and no inhibition of the development of atherosclerotic lesions [27]. The casts placed at high pressures should also eliminate pulsatile expansion of the arterial wall. Therefore, it appears that in this model the major effect of periarterial casts on the development of atherosclerotic lesions is through the localized reduction of intramural stress and not through
the accompanying alterations in pulse pressure or pressure waveform. The 8 rabbits (5 with chronic casts and 3 controls) were anesthetized with intramuscular ketamine (50 mg/kg) and acepromazine (5 mg/kg) and l-2% halothane by face mask. The femoral and carotid arteries were exposed surgically and 16-gauge catheters were placed in two femoral arteries and one carotid artery. The rabbits were then killed by intravenous injection of concentrated pentobarbital. The aorta was immediately flushed and perfused with 2.5% glutaraldehyde in Krebs-Henselyte buffer solution (pH 7.4, temperature 37°C) through the carotid catheter. After all intravascular blood had been flushed from the femoral catheters, these vessels were clamped and a hydrostatic pressure of 100 mm Hg was maintained for 2 h. The aorta was
214 then excised and the cast carefully removed. The aortic segments were further fixed in buffered 2.5% glutaraldehyde, pH 7.4 at 4 o C, for 24-48 h. The aortic segments were processed for SEM of the luminal surface. Specimens studied included the unbranched (straight) abdominal aorta, the control aorta-left renal artery branch segment as well as other aortic branch segments including the intercostal, celiac, superior mesenteric, and right renal arteries, and the casted aorta-left renal artery branch segment (Fig. 3). During fixation and pr&e&ing, care was taken to maintain the normal aortic wall curvature and to minimize any artifact due to flattening of the specimen. Each specimen was rinsed three times for ten minutes in 0.1 M Sorenson’s phosphate buffer.
The specimens were stained and fixed in 1% osmium tetroxide ‘for 1 h, then rinsed again 3 times for 10 min in 0.1 M Sorenson’s solution. The tissue was the‘n dehydrated in 40%, 60%, 80% and 100% ethanol (10 min each step), and critical-point dried for 30 min in a Tousimis CPD apparatus. The specimens were mounted on SEM stubs and sputter-coated with gold-palladium. Silver paint was used to connect the specimens to the stub. The luminal surface of each specimen was examined with a JEOL JSM-35C scanning electron microscope. Results Scanning electron micrographs of the aorta were analyzed to determine the endothelial cell
of the luminal surface of the aorta at the left renal artery ostium (A) and the celiac artery ostil dm Fig. 5. SEM photomicrographs an intimal surface that is convoluted and has frequent spindle-shaped endothel lial (B- D) of control rabbits. 5A-B demonstrate cell borders. C-D demonstrate cobblestone shaped endothelial cells. These cells are not oriented in cell s (arrow) with separated the direction of bulk blood flow. Many cells have bulging surfaces that protrude into the lumen. A-D document rather well varic ms configurations of endothelial cells seen in the branch areas.
Fig. 5. (continued).
morphology of the straight segments and branch segments in 5 casted rabbits and 3 control rabbits. The morphology of the straight (nonbranched) segment of the abdominal aorta, the control aorta-arterial branch, and the casted aorta-left renal artery branch were compared. All photomicrographs shown are 1600 X magnification and are oriented such that the direction of blood flow is approximately from the top to the bottom. All photomicrographs of the branch areas show the aortic luminal surface just distal to the central portion of the flow divider (Fig. 3). Care was taken to photograph all specimens at or near zero degree tilt angle to minimize any artifact introduced by oblique viewing of specimens. Straight segment
The intimal surface of a straight segment of the abdominal aorta is shown in Fig. 4A-B. SEM
of this area demonstrates a topographic pattern of endothelial cells having uniform size and a preferential orientation along the length of the aorta in the direction of blood flow. The endothelial cells are flat and the cell borders appear intact. No spindle-shaped cells are seen. Control branch
The intimal surface of the aorta-left renal artery branch (Fig. 5A) and the aorta-celiac artery branch (Fig. 5B) in control rabbits is shown in figures. When compared with the straight portion, the overall topographic pattern is not uniform. In some areas, the endothelial cells appear oriented with the long axis in the direction of blood flow (Fig. 5A), but in other areas they are oriented almost 45 o to the direction of flow (Fig. 5BI. The cells are less uniform in size when compared to those of the straight segment. Some
216
Fig. 5. (continued).
endothelial cells are thickened and spindleshaped. Cell borders no longer appear uniformly intact, especially in the areas occupied by spindle-shaped cells. The intima has a convoluted surface. The intimal surface of the aorta-celiac artery branch is shown in Fig. 5C-D, where there is a different morphologic pattern of the endothelial cells. The cells are rounded and disoriented with respect to blood flow (Fig. 5C). The central portions of the cells are prominent and protrude into the lumen of the artery, and have a cobblestone appearance (Fig. 5C). Fig. 5D shows endothelial cells which have nuclei that bulge into the lumen. These cells are oriented at an angle to the direction of blood flow. Similar patterns occur at the branch sites of the intercostal, superior mesenteric, and right renal arteries of control rabbits.
Casted branch
The intimal surface of a casted aorta-left renal artery branch is shown in Fig. 6A-B. The overall topographic pattern is similar to that of straight segments of the abdominal aorta (Fig. 4). The intimal surface is smooth. The endothelial cells are of uniform size and are oriented predominantly in the direction of blood flow. Individual cells are flat, the cell borders are intact, and there are no spindle-shaped cells. The morphology of endothelial cells was the same when the cast was placed for 4, 7 or 8 weeks (Fig. 6A, B). Discussion Endothelial cell morphology has been investigated extensively using SEM [16-18,30-331. Endothelial cells in straight segments of arteries
217
Fig. 5. (continued).
tend to be arranged in flat, uniform sheets with their long axes oriented in the direction of blood flow. At the site of branching, particularly along the distal flow divider of the ostium, various alterations in endothelial cells have been described including spindle shaped cells, separation of intercellular junctions, denudation of cells, and adherence of platelets [16-l&30-35]. These alterations have been attributed to increased shear stress [17,18]. Whether these changes represent true alterations in endothelial morphology or are artifacts of the fixation technique remains unresolved. Fixation and drying for SEM causes some tissue shrinkage and may produce artifacts. Artifacts are thought to be minimized when in situ perfusion fixation is used, with the fixative solution at physiologic pressure, temperature, pH, and osmolality. Even with this technique, morphologically altered endothelial cells were found
at branch sites, which most investigators believe indicates in vivo endothelial cell damage [16,18]. These altered cells are typically spindle shaped [17,18]. The separation of cell borders has also been described [16]. However, Zarins et al. demonstrated that a significant artifact was introduced by flattening the specimens and distorting the ostial flow divider [36]. They found that flattening the specimen caused cell separation and denudation, as well as spindle cell formation and that these changes did not occur if normal arterial wall curvature was carefully maintained. However, Zarins also described spindle cells at the branch site when there was minimal arterial wall distortion during fixation, and concluded this could represent endothelial cell injury [36]. Reidy, using intra-aortic casts and SEM to minimize artifacts due to tissue processing, also found morphologically altered endothelial cells (spindle
218 cells) at branch sites [19]. Recently, Reidy proposed that these cells may represent a cellular adaptation to local flow conditions rather than cell injury [21]. In the current investigation we observed a uniformly normal endothelium with flat intact cells along all straight segments of aorta. The branch sites, however, had many morphologically altered endothelial cells around the distal flow divider. The endothelial cell morphology at these branch sites displayed a morphologic spectrum ranging from spindle-shaped with disrupted cell borders to cobblestone-shaped with loss of cell orientation. Morphologically altered endothelial cells were present at branch sites in spite of our use of meticulous techniques to reduce fixation artifacts. For example, we used perfusion fixation under physiologic conditions, maintained normal
arterial wall curvature during SEM processing, and viewed all specimens near zero degree tilt angle. Our observations of normal vessels are the same as those reported by many investigators [17-19,361. Our most important observation was that there were no endothelial cells with altered morphology when intramural stress was reduced, i.e., when a rigid cast encased the arterial branch area and maintained the reduced intramural stress. The endothelium in the casted branch sites demonstrated uniform, flat topography with intact cell borders, and was identical to cells occurring in straight segments of the aorta. This finding suggests that by decreasing intramural stress the endothelial cell morphology in the branch areas has become the same as that which occurs in straight regions, where the stress is naturally lower.
of the luminal surface of the aorta at the casted left renal artery ostium. The intimal Fig. 6. (A-B) SEM photomicrographs mooth with uniform-appearing, flat, elongated endothelial cells. Cell borders are intact and the cells are oriented is z.1 direction of bulk blood flow. Cast duration is as follows: (A) 4 weeks, (B) 7 weeks.
surf ‘ace in the
219
Fig. 6. (continued),
The absence of morphologically altered endothelial cells in the casted branch sites is most likely due to reduced intramural stress and not to some adverse effect of the cast on the vessel wall produced by chemical or exothermic reactions. This is supported by our previous observation that atheroma formation is absent in cholesterolfed rabbits at casted branch sites, independent of the type of cast material used (silicone versus methyl-methacrylate dental acrylic) [27]. In addition, casts placed under hypertensive conditions, which do not cause a reduction of intramural stress, did not inhibit atheroma formation. A periarterial cast placed at a high blood pressure is just as likely to interfere with arterial innervation, or with vasovasarum, as is a cast placed at low blood pressure. Therefore, the effect of the cast on arterial innervation or on vasovasarum, if present at all, is not a significant factor in this model.
Ideally, the proper controls for the low-pressurecasted rabbits would be rabbits in which casts had been placed at a high pressure and then endothelial cell morphology compared. However, as mentioned above, our studies have shown that casts placed at high pressures did not inhibit the development of atherosclerotic lesions, implying that the condition of the artery and endothelial cells was most likely the same as in unoperated controls [27]. The relevance of these findings to the pathogenesis of atherosclerosis remains speculative. Endothelial cells normally form a continuous lining of the arterial lumen and function as a relatively impermeable barrier to plasma proteins [37,381. Several proteins (including LDL) are transported across endothelial cells, contributing to the control of the extracellular milieu of the vessel wall and providing nutrients for smooth
220 muscle cells [34,37]. Endothelial cell injury is difficult to define. Most investigators use morphologic alterations, increased endothelial cell permeability, or increased endothelial cell turnover as indicators of injury [34,35,37]. More recently, it has been proposed that functional changes may occur in injured endothelium without accompanying morphologic changes [34,35]. These include release of endothelial-derived growth factor (EDRF), release of procoagulants (thromboplastin, PGI,), and activation of platelet and monocyte adherence, all of which may contribute to the pathogenesis of atherosclerosis [351. If this argument is true, then changes in cell morphology could indicate even more profound changes in cell function. The altered morphology in the control branch area may be the consequence of a very high localized intramural stress. We [39-421 and others [431 have found that the permeability of the artery to low density lipoprotein at these locations is substantially higher than at nonbranch regions. Reduction of intramural stress by the cast changed the endothelial cell morphology. This change is most likely the adaptive response of the arterial wall to reduced stress, and, indeed, adaptive changes should also occur in smooth muscle cells. Endothelial cells of this morphology may function as a more effective barrier to plasma constituents and to cellular elements, thereby imparting antiatherogenic properties to the arterial wall. Although the changes discussed have been attributed primarily to reduced intramural stress, the elimination of pulsatile expansion of the artery or creation of slightly altered flow conditions by the cast could have had some influence on these changes. Similar adaptive changes may be taking place in patients in whom hypertension has been brought under control. In summary, the arterial branch regions have morphologically altered endothelial cells. The branch regions also have high intramural stress. Reduction of the intramural stress changes the morphology of endothelial cells in the branch region to that normally seen in the nonbranch region. Hence, arterial intramural stress appears to influence the morphology of endothelial cells. The morphology that results from a reduction of intramural stress appears to be associated with the absence of atheroma formation.
Acknowledgment The authors are grateful to Marjorie Garmey for her technical assistance, and to Jan Redick and Bonnie Sheppard for their assistance with scanning electron microscopy.
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221 L., Svendsen, E., Endothelial cell injury. In: 16 Jorgensen, Carlson L.A. (Ed.), International Conference on Atherosclerosis 1977, Raven Press, New York, 1978, p. 561. G., Bjorkerud, S., Brattsand, R., Bylock, A., 17 Bondjers, Hansson, G., Hansson, H.A., Endothelial injury in normocholesterolemic and hypercholesterolemic rabbits. In: Carlson L.A. et al. (Eds.), International Conference on Atherosclerosis 1977, Raven Press, New York, 1978, p. 567. 18 Reidy, M.A., Bowyer, D.E., Scanning electron microscopy of arteries. The morphology of aortic endothelium in hemodynamically stressed areas associated with branches. Atherosclerosis, 26 (1977) 181. 19 Reidy, M.A., Levesque, M.J., A scanning electron microscopic study of arterial endothelial cells using vascular casts. Atherosclerosis, 28 (1977) 463. between blood 20 Langille, B.L., Adamson, S.L., Relationship flow direction and endothelial cell orientation at arterial branch sites in rabbits and mice. Circ. Res., 48 (1981) 481. 21 Reidy, M.A., Langille, B.L., The effect of local blood flow patterns on endothelial cell morphology. Exp Mol. Pathol., 32 (1980) 276. 22 Levesque, M.J., Liepsch, D., Moravec, S., Nerem, R.M., Correlation of endothelial cell shape and wall shear stress in a stenosed dog aorta. Arteriosclerosis, 6 (1986) 220. 23 Thubrikar, M.J., Roskelly, S.K., Eppink, R.T., Study of stress concentration in the walls of bovine coronary arterial branch. J. Biomech., 23 (1989) 15. 24 Thubrikar, M.J., Eppink, R.T., Roskelly, S.K., Finite element stress analysis of coronary arterial branch. Proceedings of the 39th Annual Conference on Engineering in Medicine and Biology, Washington DC. The Alliance for Engineering in Medicine and Biology, 28 (1986) 249. M.J., Manuel, L., Eppink, R.T., Intramural 25 Thubrikar. stress at arterial bifurcation in vivo. Proceedings of the 40th Annual Conference on Engineering in Medicine and Biology, Washington, DC. The Alliance for Engineering in Medicine and Biology, 29 (1987) 208. M.J., Nolan, S.P., Prevention of atherosclerosis 26 Thubrikar. by reduction of arterial intramural stress in rabbits (Abstr). Fed. Proc., 46 (1987) 720. M.J.. Baker, J., Nolan, S., Inhibition of 27 Thubrikar, atherosclerosis associated with reduction of arterial intramural stress in rabbits. Arteriosclerosis, 8 (1988) 410. 28 Fluid Mechanics of Arterial Flow. In: Wolf, S., Werthessen, N.T. (Eds.), Dynamics of Arterial Flow, Plenum Press, New York, Vol. 115, 1979, p. 55. D.P., Zarins, C.K., Glagov, S., Pul29 Ku. D.N., Giddens, satile flow and atherosclerosis in the human carotid bifurcation. Arteriosclerosis. 5 (1985) 293.
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Thorgeirsson, G., Robertson, A.L., The vascular endothelium -Pathobiologic significance. Am. J. Pathol., 93 (1978) 803. Buss, H., Hollweg, H.G., Scanning electron microscopy of blood vessels: A review. Scanning Electron Micros., 2 (1977) 467. Shimamoto, T., Yamashita, Y., Numano, F.. Sunaga, T., Scanning and transmission electron microscopic observation of endothelial cells in the normal condition and in initial stages of atherosclerosis. Acta. Pathol. Japan, 21 (1971) 93. Nelson, F.C., Gertz, S.D., Rennels, M., Forbes, MS., Blaumanis O., Ultrastructural changes in the arterial endothelium: possible relationship to atherosclerosis. In: Scheinberg, P, (Ed.), Cerebrovascular Diseases. Raven Press, New York, 1976, p. 97. Ross, R., The pathogenesis of atherosclerosis - an update. N. Engl. J. Med., 314 (1986) 488. Reidy, M.A., Biology of disease, A reassessment of endothelial injury and arterial lesion formation. Lab. Invest., 53 (1985) 513. Zarins, C.K., Taylor, K.E., Bomberger, R.A., Glagov, S., Endothelial integrity at aortic ostial flow dividers. Scanning Electron Microsc. 3 (1980) 249. Ross, R., Glomset, J.A., The pathogenesis of atherosclerosis. N. Engl. J. Med., 295 (1976) 369, 420. Huttner, I., Boutet, M., More, R.H., Studies on protein passage through arterial endothelium. I. Structural correlates of permeability in rat arterial endothelium. Lab. Invest., 28 (1973) 672. Christie. A., Thubrikar, M., Holloway, P., et al., Increased low density lipoprotein permeability in ostial regions of rabbit aorta. Federation of American Societies for Experimental Biology. 73rd Annual Meeting, New Orleans cabstract). 1989, A1216. Thubrikar, M., Christie, A., Cao-Danh, H., Holloway, P., Nolan, S., Metoprolol reduces low density lipoprotein uptake in aortic regions prone to atherosclerosis. FASEB J., 4 (1990) A1151. Cao-Danh, H., Thubrikar, M., Christie, A.. Holloway, P., Nolan, S., Receptor mediated low density lipoprotein transport in aortic regions prone to atherosclerosis. FASEB J., 4 (1990) Al 152. Thubrikar, M., Christie, A.M., Cao-Danh, H., Holloway, P.W., Nolan, S.P., Mechanism of low-density-lipoprotein transport in aortic regions prone to atherosclerosis. First World Congress of Biomechanics, Univ. of California, Vol. II, 1990, pg 186. Schwenke, D.C., Carew, T.E., Initiation of atherosclerotic lesions in cholesterol-fed rabbits. Arteriosclerosis, 9 (1989) 895.
Atherosclerosis,89 (1991) 209-221 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0021.9150/91/$03.50 ADONIS
002191509100152L
ATHERO
04682
Change in endothelial cell morphology at arterial branch sites caused by a reduction of intramural stress * Joseph
W. Baker
‘, Mano J. Thubrikar ‘, Jayashri S. Parekh and Stanton P. Nolan ’
‘, Michael
S. Forbes
2
’Department ofSurgery and 2 Department of Physiology, &ice&y
of Virginia Health Sciences Center, Charlottesl,ille, Virginia 22908 (U.S.A.)
(Received 18 March, 1991) (Revised, received 9 May, 1991) (Accepted 10 May, 1991)
Summary Arterial branch sites have very high intramural stresses at physiologic intraluminal pressures; the same sites have a predilection for atherosclerosis. The effect of intramural stress on endothelial cell morphology was investigated. Five rabbits had permanent casts placed around a segment of the abdominal
aorta-left renal artery branch area during controlled hypotension, thus reducing intramural stress without narrowing the lumen. These five animals, and three normal rabbits, were sacrificed after 4-8 weeks, and the vessels were perfused with buffered 2.5% glutaraldehyde for 2 h at 100 mm Hg pressure. The aortas were examined by scanning electron microscopy. In normal aortas, the distal region of the ostia of the left renal and celiac arteries just beyond the flow divider displayed many morphologically altered endothelial cells ranging from spindle shape to cobble-stone shape. The same aortic area of casted rabbits, as well as the straight abdominal aorta in all rabbits, showed a smooth surface of endothelial cells with intact cell borders and no morphologically altered cells. At branch sites, the occurrence of morphologically altered endothelial cells may be due to increased intramural stress. When intramural stress is reduced, the morphology of branch endothelial cells changes to resemble that of the unbranched regions. In conclusion, endothelial cell morphology changes in response to changes in intramural stress.
Key words:
Endothelial
morphology;
Arterial
branch;
Intramural
stress; Atherosclerosis
Introduction * This work was presented
in part
at the 1987 and
1989
FASEB meetings. Correspondence to: Mano J. Thubrikar, Ph.D. Department of Surgery, MR4 Box 3111 University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, U.S.A.
Endothelial
cells are known to respond to a
variety of mechanical stimuli. In cell culture studies, endothelial cells align in the direction of flow,
210 i.e., the cells become elongated and oriented with their long axis parallel to the direction of flow [l-4]. Endothelial cells cultured on a compliant membrane undergoing cyclic stress become oriented perpendicular to the direction of stretching [.5-71. Endothelial cells, therefore, orient not only in response to flow but also in response to deformation of the underlying substrate. In arteries, endothelial cells are aligned along the axis of the artery [S-10]. This orientation could result from the synergistic actions of blood flow and arterial stretch. In vivo the artery undergoes stretching in the circumferential direction, which would tend to orient the cells along the length of the artery. The direction of blood flow is along the length of the artery, which would also orient the cells in that direction. In the leaflets of the aortic valve.
the endothelial cells align not in the direction of blood flow, but perpendicular to it [ll]. In this case, it was suggested that the cells are oriented in the direction of the maximum tensile stress in the leaflet [ll]. Endothelial cell injury has been implicated in the process of atherogenesis. Since atherosclerotic lesions form at sites of arterial branching in both humans [12,13] and animals [14,15], an understanding of endothelial cell morphology at these sites would be of great value. It has previously been reported that endothelial cell morphology in branching regions is different from that in straight regions [16-211 and that this difference may be due to the complex blood flow patterns occurring in localized regions of the branches [20-221. The arterial branch area is
Fig. 1. Maximum principal stress contours on the inner surface of the artery. These stresses are due to an incremental pressure loading of 40 mm Hg. The stress increases from one contour to the next towards the ostium. The stress increase occurs at both the proximal and distal regions of the ostium. The numbers represent stress values in MPa. These results were obtained from a bovine circumflex coronary arterial branch [23]. (This figure was taken from our publication, J. Biomechanics, 23;1989:15.)
211 unique in other ways as well. Intramural stresses, which are produced by the intraluminal blood pressure, are quite unevenly distributed in the branch area. Intramural stresses are tensile stresses, and they are responsible for the stretching of the artery. They are produced by blood pressure, not by blood flow, and they occur throughout the thickness of the arterial wall. We have found that intramural stresses in branch regions are 3-7 times those in nonbranch (straight) regions [23-2.51. Intramural stress is also highest on the inner surface of arteries (Fig. 1) [23]. Intramural stress is high at branch sites because the ostium of the arterial branch creates a reduction in the quantity of arterial tissue of the main artery. High intramural stresses at the branch are accompanied by significantly increased stretching (high strains) of the wall locally [23]. These high stresses and high strains should be associated with altered endothelial cell morphology at the branch site. We have observed that when intramural stress is reduced in branch areas, there is an inhibition of the development of atherosclerotic lesions [26,27]. In the present study, we investigate whether there is a difference between endothelial cell morphology in regions of high as opposed to regions of low intramural stress and whether the morphology changes when intramural stress is reduced. Methods
Endothelial cell morphology was studied with scanning electron microscopy. Intramural stress
Fig. 2. (A) Diagram of a rabbit abdominal aorta and major branches with a cast around the aorta - left renal artery segment. (B) perpendicular forces are buttressed by the rigid cast, and intramural stress is reduced. (0 parallel forces related to blood flow are not significantly altered by the cast.
Celiac A. Superior Mesenteric
Rtght Renal A
A.
Lett Kenal
A
Blood Flou
B
Fig. 3. (A) Schematic presentation of the abdominal aorta showing four major arterial branches under study. Arterial branch areas and straight segments were examined by SEM. (B) Schematic presentation of the opened aorta showing the ostium of a branch. SEM photographs were taken at the distal lip of the ostium (69) and at a straight region of the aorta ( ? ? ).
in the arterial branch area was reduced by placement of a periarterial cast. Five adult New Zealand white rabbits (body wt. 4.0-4.5 kg) were anesthetized with intramuscular ketamine (50 mg/kg) and acepromazine (5 mg/kg), and maintained on l-2% halothane by face mask. Intravenous and intra-arterial catheters were placed percutaneously in ear vessels for intravenous infusion and arterial blood pressure measurement. A midline laparotomy was performed, and a segment of the abdominal aorta and proximal left renal artery was isolated. The aorta and left renal artery were dissected free of fatty tissue circumferentially and a small piece of metallic foil was placed posterior to the branch area to form the back of the cast. The aorta-left renal artery branch was then covered with warm saline for 10 min to resolve any vasospasm. The mean arterial blood pressure was then lowered to 40 mm Hg and maintained by the intravenous infusion of nitroprusside. During this state of controlled hypotension, liquid methyl-methacrylate dental acrylic compound was poured around the isolated segment of aorta and proximal left renal artery. The cast was allowed to harden while the blood pressure was maintained at a constant level, and the nitroprusside was then discontinued. This produced a circumferential cast which encased the segment of aorta and proximal left renal artery. The rabbits underwent standard closure of the laparotomy and were allowed to recover for 4-8 weeks on a standard rabbit diet. Three control (unoperated) adult New Zealand white rabbits
Fig. 4. (A-B) SEM photomicrographs smooth. Endothelial cells have uniform direction of blood flow. A and B are noted. Note: Figs. 4-6: magnification
of the luminal surface of a straight portion of the abdominal aorta. The intimal surface is size and they are flat and elongated. Cell borders are intact and the cells are oriented in the different rabbits. A slight variation in the appearance of normal endothelial cells may be is 1600x and the direction of blood flow is approximately from top to bottom of the photomicrographs.
4.7-5.1 kg) were also used to study endothelial morphology in the normal branch area. The technique described above has been used by us previously to reduce intramural stress [27]. Intramural stress is related directly to intraarterial pressure. In the cylindrical segment of the artery
(wt.
Pr Pr uC= - and u, = 2T T where a, and (+, represent stress in the circumferential and longitudinal direction and P, R and T represent arterial pressure, radius and thickness, respectively. In branch areas, stress distribu-
tion is complex (Fig. 1) and stress is increased by 300-700% mainly as a result of the presence of a branch ostium 123-251. When blood pressure is reduced, stress becomes lower and the diameter of the artery decreases slightly, probably less than 4%. When a cast is placed at a reduced blood pressure the artery remains in this configuration and consequently in this state of decreased stress. When blood pressure is allowed to rise, the artery is prevented from expanding because of the rigid cast and a low intramural stress is maintained in the casted segment of the artery. Our previous studies showed that cast placement did not cause measurable narrowing of the arterial lumen or a decrease of arterial wall thickness [27]. Because
213
Fig. 4. (contimied)
the cast is rigid, it does eliminate pulsatile expansion of the artery; however, it is not expected to alter the regions of high and low fluid shear (Fig. 2). Most of the studies of fluid shear have been performed using rigid glass models of arteries [28,29]. It is possible that the cast may produce slight changes in pressure waveform or pulse pressure; however, the influence of these changes must be minor, because, when casts are placed at high systemic pressures (9.5 mm Hg), there is no reduction of intramural stress and no inhibition of the development of atherosclerotic lesions [27]. The casts placed at high pressures should also eliminate pulsatile expansion of the arterial wall. Therefore, it appears that in this model the major effect of periarterial casts on the development of atherosclerotic lesions is through the localized reduction of intramural stress and not through
the accompanying alterations in pulse pressure or pressure waveform. The 8 rabbits (5 with chronic casts and 3 controls) were anesthetized with intramuscular ketamine (50 mg/kg) and acepromazine (5 mg/kg) and l-2% halothane by face mask. The femoral and carotid arteries were exposed surgically and 16-gauge catheters were placed in two femoral arteries and one carotid artery. The rabbits were then killed by intravenous injection of concentrated pentobarbital. The aorta was immediately flushed and perfused with 2.5% glutaraldehyde in Krebs-Henselyte buffer solution (pH 7.4, temperature 37°C) through the carotid catheter. After all intravascular blood had been flushed from the femoral catheters, these vessels were clamped and a hydrostatic pressure of 100 mm Hg was maintained for 2 h. The aorta was
214 then excised and the cast carefully removed. The aortic segments were further fixed in buffered 2.5% glutaraldehyde, pH 7.4 at 4 o C, for 24-48 h. The aortic segments were processed for SEM of the luminal surface. Specimens studied included the unbranched (straight) abdominal aorta, the control aorta-left renal artery branch segment as well as other aortic branch segments including the intercostal, celiac, superior mesenteric, and right renal arteries, and the casted aorta-left renal artery branch segment (Fig. 3). During fixation and pr&e&ing, care was taken to maintain the normal aortic wall curvature and to minimize any artifact due to flattening of the specimen. Each specimen was rinsed three times for ten minutes in 0.1 M Sorenson’s phosphate buffer.
The specimens were stained and fixed in 1% osmium tetroxide ‘for 1 h, then rinsed again 3 times for 10 min in 0.1 M Sorenson’s solution. The tissue was the‘n dehydrated in 40%, 60%, 80% and 100% ethanol (10 min each step), and critical-point dried for 30 min in a Tousimis CPD apparatus. The specimens were mounted on SEM stubs and sputter-coated with gold-palladium. Silver paint was used to connect the specimens to the stub. The luminal surface of each specimen was examined with a JEOL JSM-35C scanning electron microscope. Results Scanning electron micrographs of the aorta were analyzed to determine the endothelial cell
of the luminal surface of the aorta at the left renal artery ostium (A) and the celiac artery ostil dm Fig. 5. SEM photomicrographs an intimal surface that is convoluted and has frequent spindle-shaped endothel lial (B- D) of control rabbits. 5A-B demonstrate cell borders. C-D demonstrate cobblestone shaped endothelial cells. These cells are not oriented in cell s (arrow) with separated the direction of bulk blood flow. Many cells have bulging surfaces that protrude into the lumen. A-D document rather well varic ms configurations of endothelial cells seen in the branch areas.
Fig. 5. (continued).
morphology of the straight segments and branch segments in 5 casted rabbits and 3 control rabbits. The morphology of the straight (nonbranched) segment of the abdominal aorta, the control aorta-arterial branch, and the casted aorta-left renal artery branch were compared. All photomicrographs shown are 1600 X magnification and are oriented such that the direction of blood flow is approximately from the top to the bottom. All photomicrographs of the branch areas show the aortic luminal surface just distal to the central portion of the flow divider (Fig. 3). Care was taken to photograph all specimens at or near zero degree tilt angle to minimize any artifact introduced by oblique viewing of specimens. Straight segment
The intimal surface of a straight segment of the abdominal aorta is shown in Fig. 4A-B. SEM
of this area demonstrates a topographic pattern of endothelial cells having uniform size and a preferential orientation along the length of the aorta in the direction of blood flow. The endothelial cells are flat and the cell borders appear intact. No spindle-shaped cells are seen. Control branch
The intimal surface of the aorta-left renal artery branch (Fig. 5A) and the aorta-celiac artery branch (Fig. 5B) in control rabbits is shown in figures. When compared with the straight portion, the overall topographic pattern is not uniform. In some areas, the endothelial cells appear oriented with the long axis in the direction of blood flow (Fig. 5A), but in other areas they are oriented almost 45 o to the direction of flow (Fig. 5BI. The cells are less uniform in size when compared to those of the straight segment. Some
216
Fig. 5. (continued).
endothelial cells are thickened and spindleshaped. Cell borders no longer appear uniformly intact, especially in the areas occupied by spindle-shaped cells. The intima has a convoluted surface. The intimal surface of the aorta-celiac artery branch is shown in Fig. 5C-D, where there is a different morphologic pattern of the endothelial cells. The cells are rounded and disoriented with respect to blood flow (Fig. 5C). The central portions of the cells are prominent and protrude into the lumen of the artery, and have a cobblestone appearance (Fig. 5C). Fig. 5D shows endothelial cells which have nuclei that bulge into the lumen. These cells are oriented at an angle to the direction of blood flow. Similar patterns occur at the branch sites of the intercostal, superior mesenteric, and right renal arteries of control rabbits.
Casted branch
The intimal surface of a casted aorta-left renal artery branch is shown in Fig. 6A-B. The overall topographic pattern is similar to that of straight segments of the abdominal aorta (Fig. 4). The intimal surface is smooth. The endothelial cells are of uniform size and are oriented predominantly in the direction of blood flow. Individual cells are flat, the cell borders are intact, and there are no spindle-shaped cells. The morphology of endothelial cells was the same when the cast was placed for 4, 7 or 8 weeks (Fig. 6A, B). Discussion Endothelial cell morphology has been investigated extensively using SEM [16-18,30-331. Endothelial cells in straight segments of arteries
217
Fig. 5. (continued).
tend to be arranged in flat, uniform sheets with their long axes oriented in the direction of blood flow. At the site of branching, particularly along the distal flow divider of the ostium, various alterations in endothelial cells have been described including spindle shaped cells, separation of intercellular junctions, denudation of cells, and adherence of platelets [16-l&30-35]. These alterations have been attributed to increased shear stress [17,18]. Whether these changes represent true alterations in endothelial morphology or are artifacts of the fixation technique remains unresolved. Fixation and drying for SEM causes some tissue shrinkage and may produce artifacts. Artifacts are thought to be minimized when in situ perfusion fixation is used, with the fixative solution at physiologic pressure, temperature, pH, and osmolality. Even with this technique, morphologically altered endothelial cells were found
at branch sites, which most investigators believe indicates in vivo endothelial cell damage [16,18]. These altered cells are typically spindle shaped [17,18]. The separation of cell borders has also been described [16]. However, Zarins et al. demonstrated that a significant artifact was introduced by flattening the specimens and distorting the ostial flow divider [36]. They found that flattening the specimen caused cell separation and denudation, as well as spindle cell formation and that these changes did not occur if normal arterial wall curvature was carefully maintained. However, Zarins also described spindle cells at the branch site when there was minimal arterial wall distortion during fixation, and concluded this could represent endothelial cell injury [36]. Reidy, using intra-aortic casts and SEM to minimize artifacts due to tissue processing, also found morphologically altered endothelial cells (spindle
218 cells) at branch sites [19]. Recently, Reidy proposed that these cells may represent a cellular adaptation to local flow conditions rather than cell injury [21]. In the current investigation we observed a uniformly normal endothelium with flat intact cells along all straight segments of aorta. The branch sites, however, had many morphologically altered endothelial cells around the distal flow divider. The endothelial cell morphology at these branch sites displayed a morphologic spectrum ranging from spindle-shaped with disrupted cell borders to cobblestone-shaped with loss of cell orientation. Morphologically altered endothelial cells were present at branch sites in spite of our use of meticulous techniques to reduce fixation artifacts. For example, we used perfusion fixation under physiologic conditions, maintained normal
arterial wall curvature during SEM processing, and viewed all specimens near zero degree tilt angle. Our observations of normal vessels are the same as those reported by many investigators [17-19,361. Our most important observation was that there were no endothelial cells with altered morphology when intramural stress was reduced, i.e., when a rigid cast encased the arterial branch area and maintained the reduced intramural stress. The endothelium in the casted branch sites demonstrated uniform, flat topography with intact cell borders, and was identical to cells occurring in straight segments of the aorta. This finding suggests that by decreasing intramural stress the endothelial cell morphology in the branch areas has become the same as that which occurs in straight regions, where the stress is naturally lower.
of the luminal surface of the aorta at the casted left renal artery ostium. The intimal Fig. 6. (A-B) SEM photomicrographs mooth with uniform-appearing, flat, elongated endothelial cells. Cell borders are intact and the cells are oriented is z.1 direction of bulk blood flow. Cast duration is as follows: (A) 4 weeks, (B) 7 weeks.
surf ‘ace in the
219
Fig. 6. (continued),
The absence of morphologically altered endothelial cells in the casted branch sites is most likely due to reduced intramural stress and not to some adverse effect of the cast on the vessel wall produced by chemical or exothermic reactions. This is supported by our previous observation that atheroma formation is absent in cholesterolfed rabbits at casted branch sites, independent of the type of cast material used (silicone versus methyl-methacrylate dental acrylic) [27]. In addition, casts placed under hypertensive conditions, which do not cause a reduction of intramural stress, did not inhibit atheroma formation. A periarterial cast placed at a high blood pressure is just as likely to interfere with arterial innervation, or with vasovasarum, as is a cast placed at low blood pressure. Therefore, the effect of the cast on arterial innervation or on vasovasarum, if present at all, is not a significant factor in this model.
Ideally, the proper controls for the low-pressurecasted rabbits would be rabbits in which casts had been placed at a high pressure and then endothelial cell morphology compared. However, as mentioned above, our studies have shown that casts placed at high pressures did not inhibit the development of atherosclerotic lesions, implying that the condition of the artery and endothelial cells was most likely the same as in unoperated controls [27]. The relevance of these findings to the pathogenesis of atherosclerosis remains speculative. Endothelial cells normally form a continuous lining of the arterial lumen and function as a relatively impermeable barrier to plasma proteins [37,381. Several proteins (including LDL) are transported across endothelial cells, contributing to the control of the extracellular milieu of the vessel wall and providing nutrients for smooth
220 muscle cells [34,37]. Endothelial cell injury is difficult to define. Most investigators use morphologic alterations, increased endothelial cell permeability, or increased endothelial cell turnover as indicators of injury [34,35,37]. More recently, it has been proposed that functional changes may occur in injured endothelium without accompanying morphologic changes [34,35]. These include release of endothelial-derived growth factor (EDRF), release of procoagulants (thromboplastin, PGI,), and activation of platelet and monocyte adherence, all of which may contribute to the pathogenesis of atherosclerosis [351. If this argument is true, then changes in cell morphology could indicate even more profound changes in cell function. The altered morphology in the control branch area may be the consequence of a very high localized intramural stress. We [39-421 and others [431 have found that the permeability of the artery to low density lipoprotein at these locations is substantially higher than at nonbranch regions. Reduction of intramural stress by the cast changed the endothelial cell morphology. This change is most likely the adaptive response of the arterial wall to reduced stress, and, indeed, adaptive changes should also occur in smooth muscle cells. Endothelial cells of this morphology may function as a more effective barrier to plasma constituents and to cellular elements, thereby imparting antiatherogenic properties to the arterial wall. Although the changes discussed have been attributed primarily to reduced intramural stress, the elimination of pulsatile expansion of the artery or creation of slightly altered flow conditions by the cast could have had some influence on these changes. Similar adaptive changes may be taking place in patients in whom hypertension has been brought under control. In summary, the arterial branch regions have morphologically altered endothelial cells. The branch regions also have high intramural stress. Reduction of the intramural stress changes the morphology of endothelial cells in the branch region to that normally seen in the nonbranch region. Hence, arterial intramural stress appears to influence the morphology of endothelial cells. The morphology that results from a reduction of intramural stress appears to be associated with the absence of atheroma formation.
Acknowledgment The authors are grateful to Marjorie Garmey for her technical assistance, and to Jan Redick and Bonnie Sheppard for their assistance with scanning electron microscopy.
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