© 1997 American Heart Association, Inc.
Articles |
Loss of Arterial Dilation in the Reendothelialized Area of the Flow-Loaded Rat Common Carotid Artery
From the Second Department of Pathology (T.S., K.K., H.N., H.M.) and the Department of Laboratory Medicine (M.S.), Akita University School of Medicine, Akita, Japan.
Correspondence and reprint requests to Dr. Tatsuo Sugiyama, the Second Department of Pathology, Akita University School of Medicine, 1-1-1 Hondo Akita 010, Japan.
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Abstract We have investigated regenerated endothelial cells and their possible contribution to arterial dilation in response to increased blood flow in rat common carotid artery (CCA). After endothelial denudation using a balloon catheter in the left CCA, an arteriovenous shunt was constructed between the left CCA and the left external jugular vein at 20 mm distal from the orifice in the denuded group. Animals that were given the arteriovenous shunt without denudation were used to form the nondenuded group. The blood flow rate in the left CCA was increased by sixfold after operation in the denuded group. We observed that endothelial cells were gradually regenerated from the orifice to the distal area and that the reendothelialized area after 4 to 8 weeks was approximately one third of the left CCA (5.31±1.49 mm at 4 weeks, 5.47±1.56 mm at 8 weeks). In the reendothelialized area of the left CCA after 4 to 8 weeks, the lumen diameter was significantly smaller than that of the nondenuded group and showed no significant difference from age-matched nonsurgical animals. The intimal and medial thickening, which would result in arterial stenosis in the reendothelialized area, was not observed in the denuded group, although the denuded control showed significant intimal thickening. From these results, we conclude that regenerated endothelial cells reduce intimal thickening but do not respond to increased blood flow to dilate the artery.
Key Words: endothelial cell • blood flow • denudation • regenerated endothelium • lumen diameter
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Introduction |
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It has been suggested that a blood vessel changes its diameter according to blood flow, dilating when blood flow increases and constricting when blood flow decreases.1 2 3 Kamiya and Togawa2 suggested that the arterial diametric changes induced by flow are due to an adaptive feedback that normalizes wall shear stress. These flow-induced arterial changes are thought to be sensed by the endothelial cells.4 5 6 7 Recently it has been suggested that regenerated endothelial cells respond differently to those preserved by various methods.8 9 10 11 12 13 Endothelial cells replicate physiologically and regenerate after injury.14 15 16 17 18 19 Furthermore, in atherosclerosis, it is believed that endothelial cells are injured and subsequently regenerate to a certain degree.20 Although hemodynamic factors appear to play an important role,21 22 it is still not clear how capable regenerated endothelial cells are in responding to blood flow change.
In this paper, we report our investigations regarding how endothelial cells regenerate after endothelial denudation and how reendothelialized areas respond to an increased blood flow in the common carotid artery of rats based on our use of a balloon catheter. Our findings show that regenerated endothelial cells lose their response to increased blood flow.
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Materials and Methods |
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Animals
A total of 225 Sprague-Dawley rats (8 weeks old, 280 to 300 g) were used. These were divided into six groups: (1) denuded group (n=57), (2) nondenuded group (n=31), (3) distended group (n=25), (4) denuded control (n=52), (5) nonsurgical group (n=25), and distended control (n=35).
The rats in the denuded group were given an endothelial denudation in the left common carotid artery (CCA) and subsequently an arteriovenous (AV) shunt operation between the left CCA and the left external jugular vein with the procedure described below. These were observed after 30 minutes, 3 days, and 1, 2, 3, 4, and 8 weeks, respectively, from the date of surgery. For each interval, at least four rats were used.
The rats in the nondenuded group were subjected to an AV shunt operation between the left CCA and the left external jugular vein without endothelial denudation, and they were used as a control. These were also observed after 1, 2, 3, 4, and 8 weeks, respectively, from the date of surgery. Tohda et al4 have previously performed precise and extensive experiments on this group. We conducted the same experiments as those reported by Tohda et al on our control group. For each interval, at least five rats were used.
The rats in the distended group were used to investigate whether the damage of media by hyperdistension disturbs the arterial dilation induced by increased blood flow. These were examined after 30 minutes, 3 days, and 1, 2, and 4 weeks, respectively, from the date of surgery. For each interval, at least five rats were used.
The rats in the denuded control were given an endothelial denudation in the left CCA without an AV shunt operation. These were examined after 30 minutes and 1, 2, 3, 4, and 8 weeks, respectively, from the date of surgery. For each interval, at least five rats were used.
The rats in the nonsurgical group were used as the age-matched group. The blood flow in the right CCA after making the AV shunt in the left CCA increased approximately by twofold,4 therefore, we did not use the contralateral artery as a nonsurgical control. They were sacrificed at 9, 10, 11, 12, and 16 weeks of age, respectively. For each interval, at least five rats were used.
The rats in the distended control were given hyperdistention in the left CCA without an AV shunt operation. These were examined after 30 minutes and 1, 2, and 4 weeks, respectively, from the date of surgery. For each interval, at least five rats were used.
Before surgery, animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The experimental methods in this article were previously approved by the Animal Research Committee, Akita University School of Medicine, Akita, Japan. All subsequent animal experiments were performed following the animal experimentation guidelines of Akita University.
Denudation of Endothelial Cells
A 2-French balloon catheter (model Fogarty Arterial
Embolectomy Catheters 2Fr, Baxter Co., Santa Ana, Calif), was intubated
into the left CCA and was inflated with room air, instead of saline.
The amount of air, inflated with a syringe (1-mL volume, Top Co.,
Tokyo, Japan), had been determined to be 0.12 mL for expansion of the
balloon to 2 mm in diameter. This size was nearly twice as large
as the normal left CCA in diameter. The catheter was inserted into the
left CCA from a longitudinal incision (1 mm in length) at 20
mm distal from the orifice. It was moved up to the orifice and after
inflation, it was pulled back slowly to the incision. It was again
pushed to the orifice and pulled back to the incision. When the balloon
was inflated, the left CCA was slightly expanded but it was not more
than 1.5 times its original control state diameter. Our preliminary
histological and scanning electron microscopical
examinations in five rats had shown that endothelial
cells were completely denuded from the orifice to the incision by the
procedure described above. The transmission electron microscopical
examination 1 day after the procedure had shown that the percentage of
degenerated and necrotic smooth muscle cells (medial necrosis rate) was
16.1±3.1% (n=4).
Hyperdistension
The arterial hyperdistension was undertaken in the
left CCA by using the same balloon catheter that was used in the
following procedure of denudation. The balloon was inflated with saline
instead of air to ensure tightness. Before introducing the catheter,
the amount of injected saline had been determined to be 0.15 mL for
expansion of the balloon to 2 to 3 mm in diameter, which was
larger than the one with air. The catheter was inserted into the left
CCA from the incision as the denuded case and was settled at the
orifice. The balloon was inflated with the predetermined amount of
saline for 10 seconds. At this time, the diameter of the left CCA
became more than 1.5 times larger than before the balloon was inserted.
After deflation of the balloon, the catheter was moved about 2 mm
and was again inflated for 10 seconds. This procedure was continued
from the orifice to the incision. The whole procedure was performed
twice. Our preliminary scanning electron microscopical observations, in
the four rats used in the procedure described above, had revealed small
patches of endothelial desquamation. We had calculated
the desquamation rate of the surface by digital image-analyzer
system (Cosmozone 2, Nikon Co., Tokyo) at 28.9±7.9% (n=4) from the
scanning electron microscopical photomicrographs (x200). However,
within 2 weeks, they were completely covered with
endothelial cells in the proximal area near the
orifice. The transmission electron microscopical examination had shown
that the medial necrosis rate was 36.8±8.5% (n=4) 1 day after
the procedure.
AV Shunt Operation
A side-to-side AV shunt was made between the left CCA and the
left external jugular vein at 20 mm distal from the orifice of the
left CCA using a stereoscopic microscope (model OMS-60, Topcon, Tokyo).
The operative procedure was performed according to the method described
by Tohda et al.4 In the denuded group and the distended
group, the incision to the left CCA, which had been used for the
catheter insertion, was then used for the AV shunt operation. In the
denuded and distended controls, the incision to the left CCA was closed
with six stitches of microsurgical suture (10-0 nylon).
Blood Flow Measurement
The blood flow rate in the left CCA was measured at 5 mm
proximal from the AV shunt with an electromagnetic flowmeter (model
MFV-3200, Nihon Koden Co., Tokyo) with a 0.5-mm lumen probe (model
Fi-005T, Nihon Koden Co) before the operation, soon after the
operation, and before the sacrifice.
Perfusion Fixation and Preparation of Tissue
After measuring the blood flow rate in the left CCA, we opened
the abdominal cavity and cannulated a catheter into the abdominal
aorta. A drainage was made at the right renal vein and after flushing
the blood with heparinized lactated Ringer's solution (50 ml), 75 ml
of 3% glutaraldehyde solution in sodium cacodylate
buffer (pH 7.4) was injected via the catheter at a controlled pressure
of 100 mm Hg under simultaneous pressure monitoring.
Then after at least 3 minutes perfusion, the left CCA was removed and
postfixed in the same fixative for at least 24 hours at 4°C.
For histology, we made successive cross-sections of the left CCA following the method described by Tohda et al.4 The complete serial sections, each 5.0 µm thick, were made from the orifice to the AV shunt. Each 100th and 101st section was stained with Masson's elastica stain and hematoxylin and eosin stain.
For scanning electron microscopy after dehydration through graded alcohols and critical point-drying, the dried vessel was cut longitudinally to obtain two half-cylinder shapes and then in the middle to make four segments. These samples were mounted and sputter-coated with gold-platinum. The surface of endothelial cells was observed using a scanning electron microscope (model JMS-T200, JEOL Co., Tokyo).
For transmission electron microscopy, the left CCA was serially sectioned, with each section 1 mm long, from the orifice to the AV shunt and then postfixed with a solution of 1% osmium tetroxide in phosphate buffer for 1 hour at 4°C. Next they were embedded in Epon, and ultrathin longitudinal sections were made. They were stained with lead citrate and uranyl acetate and were observed using a transmission electron microscope (model LEM2000, Akashi Co, Tokyo).
Morphometric Data
The first cross-section of every 100 continuous sections of the
left CCA, which was stained with Masson's elastica stain, was selected
for morphometric measurement. In other words, a total of 32 sections
were taken, each one at every 0.625 mm (5 µm x
100 x 1.25, with 1.25 as a correcting factor for tissue
shrinkage3 4 23 ; and sections were numbered from numbers 1
to 32, corresponding to the position from the orifice to the AV
shunt.
In the denuded group, the length of area from the orifice to the borderline in which the endothelium was observed was measured from section numbers 1 to 32. In the borderline between the endothelium desquamated and preserved areas, we considered the section as "endothelium covered" when more than half of the lumen in the section was covered with endothelial cells. The length of covered area was calculated from the section number (section number x 0.625 mm).
The length of regenerated areas in the denuded group and the denuded control after 2 to 8 weeks was also measured from the scanning electron microscopy photomicrographs (x150) because in some situations it was difficult to clearly distinguish the endothelial cells from the regenerated smooth muscle cells of intima even by histological sections in the denuded control. A whole panoramic photograph of the left CCA was made by piecing serial scanning electron microscopy photomicrographs together, and the length of regenerated areas was measured from the orifice to the borderline between the endothelium desquamated and preserved areas (n=3).
Measurements of the internal diameter and wall thicknesses (intima and media) were performed, the method described by Tohda et al,4 on each group after 4 to 8 weeks from operation, respectively. Sample size (n) for the measurements of vessel diameter and arterial wall thicknesses was four in each group. First, the arterial cross-section was magnified (x200) with a profile projector (model V-12A, Nikon Co). Next, the enlarged profiles of the lumen, intima, and media were traced on tracing paper. Finally, the lumen circumference and arterial wall thicknesses on the histological section were obtained by use of a digital image-analyzer system. In situ internal diameter and wall thicknesses were calculated after with correcting tissue shrinkage.3 4 23
Endothelial Cell Density
The endothelial cell density was measured from
scanning electron microscopy microphotographs (x1,000) at a position 3
to 4 mm distal from the orifice in the denuded group (n=3) and
nonsurgical group (n=3) after 4 to 8 weeks. The average of
endothelial cells per square millimeter was counted
from five microphotographs taken of each specimen.
Statistical Analysis
All of the morphometric data is represented as a
mean±SD. Statistical analyses were performed by
analysis of variance followed by Scheffe's test for multiple
comparisons to compare the results of each group and interval.
Differences were determined to be significant when the probability
value was less than .05.
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Results |
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Blood Flow Rate
In the denuded group, the blood flow rate in the left CCA significantly increased soon after the AV shunt operation by fivefold compared with the blood flow rate before making the AV shunt (P<.001) (Table 1
). The blood
flow rate gradually increased after 1 week up to 8 weeks by
approximately sixfold compared with the blood flow rate before the
operation (P<.001). The blood flow rate in the nondenuded
group also significantly increased soon after the operation by sixfold
compared with the blood flow rate before the operation
(P<.001). The blood flow rate gradually increased by
approximately sevenfold after 1 to 8 weeks compared with before the
operation (P<.001). The blood flow rate in the denuded
group was less than that in the nondenuded group after the operation
and at each interval; however, there was no significant difference
between the two groups. The blood flow rate in the distended group
increased soon after the AV shunt operation and gradually increased
after 1 to 4 weeks by approximately sevenfold compared with before the
operation (P<.001).
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Endothelium-Covered Area in the Orifice and the
Proximal Portion of the Left Common Carotid Artery
Area From the Orifice
In the left CCA in the denuded group 30 minutes after the
operation, the endothelial cells were completely
denuded from the orifice to the AV shunt (Figs 1 and 2).
From 1 week after the operation, the endothelial cells
were observed near the orifice and in the proximal portion of the left
CCA. They were continuous from the aorta. The length of the
endothelium-covered area from the orifice to the margin
of endothelial presence enlarged gradually, and the
endothelial cells were observed in approximately one
third of the proximal left CCA after 8 weeks.
Endothelial cells were not observed in two thirds of
the distal area of the left CCA.
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The length of the endothelium-covered area in the denuded control was more than one third of the proximal left CCA and was significantly larger than that in the denuded group after 2, 4, and 8 weeks, respectively (P<.01). The endothelial regeneration that was upstream from the distal was observed in the denuded control, but its length was within 5 mm.
In the left CCA in the nondenuded group after 4 to 8 weeks and in the distended group after 4 weeks, endothelial cells were observed in two thirds of the proximal area (12.5 mm long; numbers 1 to 20). There were no endothelial cells in one third of the distal area (numbers 21 to 32).4
Morphology of Endothelial Cells
Four weeks after the shunt operation, the
endothelial cells near the orifice had bulged into the
lumen and had elongated along the longitudinal axis in the direction of
blood flow (Figs 3A, 4A, and 5A). The average width of the
endothelial cells was approximately 4 µm.
Endothelial cells were packed together densely, and
cell density was significantly greater than that in the nonsurgical
group (P<.001) (Table 2). In
the cytoplasm, a thick bundle of stress fibers was observed (Fig 5B).
At the border, between the endothelium-covered and
noncovered areas, the endothelial cells were smaller
and their arrangement was irregular (Fig 4B).
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Eight weeks after the operation, the endothelial cells
near the orifice became flatter and wider compared with those after 4
weeks (Figs 3B
, 6A, and 7A). The cell density was greater than
that in the nonsurgical group (P<.001) but less than that
in the denuded group after 4 weeks (P<.01) (Table 2). The average width of the
endothelial cells was approximately 6 µm. The
surface was irregular and wrinkled, and the nuclear portion was
slightly bulged. The bundle of stress fibers was observed in the
cytoplasm but was thinner than that after 4 weeks (Fig 7B). At the
border between the endothelium-covered and noncovered
areas, the endothelial cells were smaller and their
arrangement was slightly irregular (Fig 6B), yet not as much as those
observed after 4 weeks.
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Internal Diameters
Four Weeks After the Operation
As can be seen in Fig 8, the
internal diameter in the denuded group, in two thirds of the proximal
left CCA (numbers 3 to 18, 11.25 mm from orifice), was
significantly smaller than that in the nondenuded group
(P<.05) and that in the distended group
(P<.05). There was no significant difference between the
internal diameter in the denuded group and that in the nonsurgical
group in all the sections (numbers 3 to 30). The internal diameter in
the denuded group was slightly greater than that in the denuded
control. In the nondenuded group, the internal diameter of two thirds
of the proximal left CCA (numbers 3 to 20), where
endothelial cells were preserved, was greater than that
in the nonsurgical group (P<.05). The internal diameter in
the distended group was significantly greater than that in the
distended control (P<.05) and was slightly greater than
that in the nondenuded group.
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Eight Weeks After the Operation
As can be seen in Fig 9, the
internal diameter in two thirds of the proximal left CCA (numbers 3 to
19, 12 mm from orifice) was significantly smaller than that in the
nondenuded group (P<.05), and there was no significant
difference between the internal diameter in the denuded group and that
in the nonsurgical group in all the sections (numbers 3 to 30). The
internal diameter in the denuded group was slightly greater than
that in the denuded control. In the nondenuded group, the internal
diameter in two thirds of the proximal area (numbers 3 to 20), where
endothelial cells were preserved, was further enlarged
and greater than that in the nonsurgical group (numbers 3 to 18,
P<.05).4
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Intimal and Medial Thickness
The intimal thickening was observed in the denuded control at 4
and 8 weeks and in the distended control at 4 weeks (Tables 3 and 4,
Fig 10). In the denuded control, the
intima, where endothelium existed, was significantly
smaller than that where endothelium was desquamated
(P<.05). In other groups, the significant intimal
thickening was not measured from the histological
sections. Transmission electron microscopy photomicrographs showed no
significant intimal thickening in the regenerated area of the denuded
group. In the endothelial desquamated area of the
denuded group, slight intimal thickening was partially observed, but it
was not as thick as the denuded control. There was no significant
difference in the medial thicknesses between the denuded group and the
denuded control at 4 and 8 weeks, respectively.
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Discussion |
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After denudation, the endothelial cells rapidly regenerate into the desquamated area and cover the wound. When the wound area is not so wide, endothelial cells may migrate and spread into the area from endothelium-preserved sites.17 18 However, when the wound area is wide or long, endothelial cells may not fully cover the desquamated area again.15 16 17 24
In this study, endothelial cells were completely denuded by the balloon catheter from the orifice to a portion 20 mm distal to it in the left CCA in the denuded group. After denudation, the length of the endothelium-covered area was gradually elongated from the orifice. It reached about 5 mm as early as 3 weeks after denudation; however, it gradually ceased to elongate and remained around 5 to 6 mm even after 8 weeks. The characteristics of the endothelial cells near the orifice and in the border between the endothelium-covered and noncovered areas were bulging of nucleus, increased number of cells, and irregular arrangements on the border. These endothelial cells regenerated continuously (ie, reendothelialized) from the aorta. However, it is not clear why the endothelial cell regeneration appears to have stopped to a certain extent.16 18 19 20 24 Langille et al25 have suggested that the inhibition of repair after endothelial injury under high blood flow is caused by the endothelial dysfunction secondary to hemodynamic injury. Our findings, that the regenerated area under normal flow was larger than that under high flow, may also support this suggestion. We have found that the endothelial cells change their shape after 4 to 8 weeks. After 4 weeks, the regenerated cells are elongated along the blood flow axis and are densely packed together. After 4 more weeks, the shape of the regenerated endothelial cells at 8 weeks becomes wider and the cell density decreases. It is reported that the shape of the regenerated endothelial cells is different at each interval in the rat aorta after the denudation of endothelial cells.15 17 In the present study, the morphological changes in the regenerated endothelial cells are observed not only in their shape, but also in the bundle of stress fibers in their cytoplasm. The bundle of stress fibers has been reported in the endothelial cells under high wall shear stress.26 27 The thick bundle in the regenerated endothelial cells was observed after 4 weeks, and it became thinner after 8 weeks even though the increased blood flow was continuously loaded to the left CCA. From the changes of cell density and cell shapes, we may suppose that the endothelial cells can tolerate and adjust to the increased blood flow after some period. These reendothelializations should also be present in the distal AV shunt side; however, we did not observe the upstream directed reendothelialization in any animal in the denuded group. This may be due partially to the fact that the blood flow was too strong for the endothelial cells to regenerate from the AV shunt side backward against the increased blood flow. The endothelial desquamation was shown in the distal segment of the left CCA near the AV shunt in the nondenuded group. We suggest that not surgical trauma but the very high wall shear stress was one of the main causes of endothelial desquamation.4
The significant prevention of intimal thickening in the reendothelialized segment in the denuded group (high wall shear stress group) as compared with the denuded control (normal wall shear stress group) was demonstrated in our study. The slight intimal thickening was partially observed by transmission electron microscopy in the endothelial desquamated segment in the denuded group, but its amount was not as much as that in the denuded control. Recently it has become clear that the high wall shear stress prevents intimal thickening.5 21 23 28 29 30 31 In addition, high wall shear stress is known to induce acute reduction of endothelial platelet-derived growth factor B chain mRNA in vitro.32 These results point to the effect of fluid shear stress acting on the endothelial cells as an important determinant of intimal thickening. In this respect, the regenerated endothelial cells might prevent the intimal thickening under high wall shear stress.
An artery changes its diameter depending on blood flow.1 2 3 4 5 6 7 It is thought that this phenomenon is the adaptive reaction of an artery induced by the wall shear stress caused by blood flow.2 33 It is known that the endothelial cell changes its shape when there is a change in the wall shear stress.26 27 34 35 36 37 38 39 Therefore, endothelial cells are thought to have a function of detecting the change of blood flow and changing the arterial diameter.3 4 5 6 7 In our study, the lumen diameter in the reendothelialized area of the denuded group at 8 weeks showed no change, although it is clear that the increased blood flow was loaded on the reendothelialized area for at least 4 weeks during the period of 4 to 8 weeks. However, the intimal thickening that is usually the cause of arterial stenosis was not observed. Therefore, arterial dilation was not shown in the reendothelialized area. In other words, there was no diameter response to the increased blood flow in the reendothelialized area.
In the distended group after 4 weeks, the artery where the media was temporally damaged by hyperdistension, with little endothelial denudation, showed adaptive dilation induced by the increased blood flow. This proves that even when the media has been damaged with endothelial preservation, the artery shows adaptive dilation induced by the increased blood flow. Therefore, we suggest that a loss of dilation in the reendothelialized area is caused by the dysfunction in the regenerated endothelial cells.
It has been reported that the responsiveness to some vasomotor agents in the reendothelialized artery is different from normal responsiveness.8 9 10 11 12 13 The reendothelialized artery has a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin in the porcine coronary arteries, and the lack of responsiveness is the cause of the endothelial dysfunction.8 9 The abnormal endothelium-dependent function is shown in the production, not only of constricting factor, but also of relaxing factor in the regenerated area.10 The production of an endothelium-derived relaxing factor is decreased in the regenerated endothelial cell of the rabbit carotid artery, which indicates a dysfunction in the regenerated endothelial cell.11 There is a persistent attenuation of receptor- and nonreceptor-mediated endothelium-dependent relaxations in the regenerated endothelium, which is enhanced by the hypercholesterolemia.12 13 However, recently Jamal et al40 have shown that the diameter reductions induced by decreased blood flow are preserved in the reendothelialized area of the rabbit carotid artery.
Our present results on the function of the regenerated endothelial cells are not consistent with remodeling activities, and the dysfunction might be caused by the hemodynamic injury of high blood flow.25 In this respect, we need additional studies on the flow-loaded artery for a longer period of time after denudation. The blood flow influences the endothelial cells directly and produces vasomotor agents,41 42 43 44 45 increasing vasomotor mRNA levels46 in vitro. However, it is unknown whether vasomotor action would stimulate the arterial remodeling. Recently, it has been reported that the inhibitor of nitric oxide synthesis, NG-nitro-L-arginine, inhibits the arterial dilation induced by the blood flow, and it has been suggested that the endothelial nitric oxide synthesis is involved in flow-induced dilation.47 However, it is not clear how the endothelial cells control the size of the artery, therefore additional studies focused on these points are needed.
As we have shown in this study, regeneration is not limitless, and the function of regenerated endothelial cells is not normal, ie, they fail to dilate the artery while they can respond to control intimal thickening under high wall shear stress. We recommend future studies to carefully investigate the artery and consider the morphological and functional limitations of regenerated endothelial cells.
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Acknowledgments |
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This study was supported by a grant from the Study Group on Intractable Angiitis of the Ministry of Welfare, Japan, and a grant-in aid of scientific research on a priority area of Biomechanics of Structure and Function of Living Cells, Tissue, and Organs from the Ministry of Education, Science, and Culture, Japan in 1994.
Received June 14, 1995; accepted February 26, 1997.
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