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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2891-2896.)
© 1997 American Heart Association, Inc.

Articles

Evidence for the Migration of Rat Aortic Endothelial Cells Toward the Heart

W. B. Kiosses; N. H. McKee; ; V.I. Kalnins

From the Department of Anatomy and Cell Biology (W.B.K., V.I.K.) and the Department of Surgery (N.H.M), University of Toronto, Toronto, Ontario, Canada.

Correspondence to Dr V.I. Kalnins, Department of Anatomy and Cell Biology, Medical Sciences Building, 8 Taddle Cr Rd, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. E-mail vitauts.kalnins{at}utoronto.ca

	Abstract

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Abstract Most vascular endothelial cells at the edge of experimentally induced wounds have their centrosomes oriented toward the wound in the direction of cell migration. The finding that the centrosomes in endothelial cells of nonwounded aorta and vena cava are also oriented toward the heart suggested the hypothesis that endothelial cells are normally migrating in this direction. To test this hypothesis, endothelial cells in a segment of the rat abdominal aorta were labeled with a relatively nontoxic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and the position of the labeled cells was determined 3 and 6 weeks later. The results obtained showed that in 6 of the 9 rat aortas examined at 3 weeks and 15 of the 20 rat aortas examined at 6 weeks, DiI-labeled endothelial cells had migrated various distances up to 5000 µm toward the heart. In contrast, no migration of endothelial cells was detected at the opposite end of the labeled segment, in the direction away from the heart. These results demonstrate that vascular endothelial cells in the abdominal aorta of the rat are not stationary but are migrating toward the heart. The significance of the migration of endothelial cells toward the heart is presently unknown; however, it would be interesting to explore whether or not the impairment of this migration may contribute to disease processes in which the ability to maintain an intact and normally functioning endothelial cell lining is compromised as in atherosclerosis.

Key Words: migration • endothelial cells • rats • aorta

	Introduction

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The endothelium that lines blood vessels is believed to consist of a stationary layer of cells that can be stimulated to migrate only after denudation of the endothelial cell (EC) lining.^{1 2 3 4 5} In experimentally wounded monolayers in cell culture,⁶ including vascular EC cultures,^{7 8 9} wounded endothelium in aortic organ cultures,¹⁰ and blood vessels in situ,^{5 11} most cells have their centrosomes oriented toward the wound in the direction of cell migration. The observation that centrosomes in ECs of nonwounded aorta and vena cava of pigs,¹² young rabbits,¹³ and rats¹⁴ are preferentially oriented toward the heart suggests that ECs may be normally migrating in this direction.¹²

To determine whether the aortic ECs in situ are migrating toward the heart, a segment of rat abdominal aorta was ligated and filled with a solution of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) to label the ECs. DiI is a widely used fluorescent cell marker that is relatively nontoxic to the marked cells, does not pass from marked to unmarked cells, and remains incorporated in the cell membrane,^{15 16 17} allowing the identification of marked cells for periods of up to 9 months.¹⁸ After labeling the ECs, the location of the labeled ECs was determined 3 and 6 weeks later by viewing fixed whole-mount preparations of the vessels with a scanning laser confocal microscope. Using this approach, the migration of DiI-labeled ECs toward the heart was demonstrated in the majority of the rats.

	Methods

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Animals
Forty 200-g Sprague-Dawley rats, obtained from Charles River Canada (St Constant, Quebec; 29 experimental and 11 controls), were used for this study. Husbandry was provided by the Division of Comparative Medicine at the University of Toronto according to the standards of the Canadian Council on Animal Care.

In Situ Labeling
After the rats were anesthetized with an intramuscular injection of xylazine (20 mg/mL) and ketamine (100 mg/mL; 1:1 ratio: 0.2 cc per 100 g), a 0.5-cm segment of abdominal aorta, below the renal and above the iliolumbar branches, was cleared free of surrounding tissue, ligated, and injected with 40 µg/mL of DiI (Molecular Probes), which was dissolved first in 100% ethanol¹⁵ and then diluted in medium 199 (GIBCO), to mark the ECs. The position of the proximal (nearest to heart) and distal (nearest to bifurcation of common iliacs) ligatures was marked with 10-0 nylon sutures on the adventitial surface of the vessel placed within 400 µm just outside both ligatures to indicate the boundary between the regions containing labeled and unlabeled ECs. The 33-gauge needle (Fine Scientific Tools) was introduced into the vessel lumen at an acute angle in a distal-to-proximal direction with the syringe held over the pelvis. The injection site was always closer to the distal ligature to ensure that there was enough room for the entire needle within the vessel lumen. The aortic ECs were exposed to the dye for 10 minutes, after which the DiI was aspirated and the ligatures were removed. The hole created by the needle was stitched with 10-0 nylon suture and the abdominal wound was then closed. The distance between the ligatures and this suture was measured and recorded before and after each experiment to confirm that the position of ligatures remained stationary.

Tissue Preparation and Microscopy
Immediately (n=7), 24 hours (n=4), 3 weeks (n=9), and 6 weeks (n=20) after labeling, the rats were anesthetized with an intramuscular injection of xylazine/ketamine cocktail. The abdominal and thoracic parts of the aorta were removed, cut open longitudinally, fixed in 3% paraformaldehyde, and labeled with a nuclear stain, Yoyo (ICN Immunobiologicals), to distinguish EC from smooth muscle cell nuclei, which have different orientations. The whole mounts of the aorta were then examined with an MRC 600 Biorad scanning confocal microscope (Bio-Rad) to determine the position of the DiI-labeled ECs in relation to the proximal and distal nylon sutures.

Data Acquisition
The minimum distance (immediately and 24 hours after DiI labeling) and the maximum distance (3 weeks and 6 weeks after DiI labeling) between the proximal suture and the location of DiI-labeled ECs in each vessel were measured on computer screen directly from stored confocal images using Bio-Rad data analysis software. The distances obtained in this manner were also confirmed as follows: Thermal prints of the double-labeled confocal images were made and then used to assemble a 100x magnified final image of the labeled area. This final image was assembled from approximately 70 thermal prints showing the distribution of DiI-labeled ECs and the corresponding 70 thermal prints showing the distribution of Yoyo-labeled ECs. Measurements of the distance that the labeled ECs had migrated past the proximal suture were then made directly from this image. After placing a 10-mm ² grid transparency over both final images, it was also possible to construct a map indicating where ECs were lost, unlabeled, or DiI labeled, along the aortic segment. Each square of the grid was scored as having either no ECs, unlabeled ECs, or DiI-labeled ECs, if within that square >50% of the surface fit the appropriate category. Two of these maps (one control and one experimental) were photographed on 35-mm Tmax film, and the film was then scanned using a Polaroid Sprint Scan 35. The image obtained was then imported into the Corel Draw 5 software program, where it was used to outline the region occupied by DiI-labeled ECs and regions of cell loss.

	Results

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Seven rats were examined immediately and four were examined 24 hours after exposure to DiI to determine the pattern of initial labeling of ECs within the ligated segment of aorta. Twenty-four hours was selected to allow enough time for ECs that may have been damaged by the surgical procedure to be sloughed off by the blood flow. To identify the regions in which ECs were still present after DiI labeling, the aortic segments were double labeled with Yoyo to distinguish EC nuclei, which are oriented parallel to blood flow, from smooth muscle cell nuclei, which are oriented perpendicular to flow.

In the control rats immediately and 24 hours after DiI labeling, the marked ECs were observed only between the two suture lines and formed an irregular pattern. Detailed analysis of a reconstructed image of this pattern from a rat (labeled with DiI 24 hours earlier) showed that the DiI-labeled cells covered approximately 37% of the surface area and were predominantly located near the central part of the segment in the vicinity of the injection site (Fig 1a). In the remainder of the segment, closer to the ligations, there were areas of EC loss and unlabeled ECs, which accounted for approximately 11% and 52% of the surface area, respectively. The EC loss was largely restricted to the proximal and distal suture lines, where denuded areas approximately 500 µm in width immediately and 300 µm in width 24 hours after DiI labeling were observed and likely resulted from both the manipulation of the vessel during surgery and, later, handling of the vessel for staining and mounting. It is likely that areas remained unlabeled because air bubbles trapped in the injected segment prevented even exposure and labeling of the endothelium with DiI. This scenario affected the degree of labeling and not the degree of cell loss, which remained low. In fact, in the control ligated segments filled with DiI, where air bubbles were evident during the labeling procedure, the labeling of the endothelium was very sparse. Increasing the incubation time with DiI from 10 to 20 and 30 minutes did not appear to increase the number of labeled ECs, nor did it the change the pattern of labeling. Thus, the loss of some ECs and our inability to label all the remaining ECs within the ligated segment both contributed to the lack of labeling near the suture lines observed at 0 and 24 hours (Figs 1a and 2). There was no detectable loss of ECs from regions of the abdominal aorta outside the labeled segment at 0 and 24 hours, as revealed by the presence of EC nuclei stained with Yoyo in these regions.

View larger version (53K): [in this window] [in a new window]   Figure 1. Distribution of labeled ECs (gray) in a control rat aorta 24 hours after labeling (a) and in an experimental rat aorta 3 weeks after DiI labeling (b). The labeling pattern at both 24 hours and 3 weeks is irregular; however, labeled ECs at 24 hours are found only between the two suture marks, whereas labeled ECs at 3 weeks are found beyond the proximal suture, toward the heart. The DiI-labeled ECs within the rectangular boxes (a, top and b, bottom) are shown in Figs 2 and 3, respectively. At 24 hours and 3 weeks, it was difficult to distinguish whether ECs were lost, unlabeled, or DiI labeled in the black region because of the seepage of the dye into the subintimal and medial cell layers due to injury to the endothelium. The original injection site is marked with a circle. Bars=500 µm.

View larger version (45K): [in this window] [in a new window]   Figure 2. Micrograph of a portion of a whole mount of abdominal aorta, (box in Fig 1a) indicating the distribution of ECs labeled with DiI 24 hours earlier. DiI-labeled ECs are present only on the distal side (left) of the proximal suture in this control rat. Bar=250 µm.

In aortas examined 3 and 6 weeks after labeling, DiI-labeled ECs were observed extending beyond the proximal suture, the suture nearest to the heart in 6 of the 9 rat aortas examined after 3 weeks, including the one subjected to detailed analysis (Figs 1b and 3) and in 15 of the 20 rat aortas examined after 6 weeks. In the remaining 3 rat aortas examined after 3 weeks and 5 rat aortas examined after 6 weeks, no migration of DiI-labeled ECs beyond the proximal suture was observed.

View larger version (51K): [in this window] [in a new window]   Figure 3. Micrograph of a portion of a whole mount of abdominal aorta (box in Fig 1b) indicating that DiI-labeled ECs have migrated beyond the proximal suture, in the direction of the heart. Some labeled ECs near the proximal suture are obstructed by marker dye used to indicate the position of the nylon suture. Bar=250 µm.

There was considerable variability in the distance (ranging up to 1000 µm) that the DiI-labeled ECs were observed distal to the proximal suture line immediately and 24 hours after DiI labeling between different rat aortas (Fig 4) and along the circumference of the aortic wall within the same rat (Fig 1a). As a consequence, there was also considerable variability in the distance (ranging up to 5000 µm) that the DiI-labeled ECs were found beyond the proximal suture, toward the heart, between different rat aortas (Fig 4) and along the circumference of the aortic wall within the same rat (Fig 1b).

View larger version (19K): [in this window] [in a new window]   Figure 4. Graph showing the minimum distance (µm) between the proximal suture and the DiI-labeled ECs observed at 0 and 24 hours after labeling and the maximum distance (µm) that the DiI-labeled rat aortic ECs had migrated beyond the proximal suture, in 6 of the 9 rats after 3 weeks and in 15 of the 20 rats after 6 weeks. No migration of labeled ECs was detected past the distal suture at the opposite end of the labeled segment.

In the 3 control and 9 experimental rat abdominal aortas that were examined in detail, the length (along the longitudinal axis of the vessel) of the region with DiI-labeled ECs was never longer than the distance between the two suture markers. Therefore, it is unlikely that the DiI-labeled ECs had migrated in both directions. Furthermore, labeled ECs were never detected beyond the distal suture at the opposite end of the segment, ie, in the direction away from the heart, in any of the 29 rat aortas examined.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In summary, we observed that some DiI-labeled ECs of the rat abdominal aorta had migrated beyond the proximal suture in the direction of the heart 3 and 6 weeks after labeling and that none were detected past the distal suture at the opposite end of the labeled segment. This finding indicates that ECs of the abdominal aorta of the rat are not stationary but normally migrating toward the heart.

Since we do not know the precise location of the DiI-labeled cells behind the proximal suture initially, the measured distance that the ECs migrated beyond the proximal suture is probably a fraction of the total distance they had migrated after labeling. In rats in which the labeled ECs initially were furthest away from the proximal suture, there may not have been enough time for ECs to migrate beyond this suture within the 3- and 6-week periods. It is very likely that this factor may account for the instances in which migration was not detected beyond the proximal suture, ie, in 28% of the aortas examined. Manipulation of these vessels during surgery may also have caused more extensive EC denudation that had to be repaired before migration of ECs toward the heart could continue.

Generally, as a result of the mechanical manipulation during the surgical procedure and ligation of the vessel, there was some damage to the endothelium and some cell loss. This loss of ECs at 24 hours after labeling accounted for <11% of the cells and was largely restricted to the proximal and distal suture lines, where denuded areas approximately 300 µm in width were observed. Such regions of cell loss were wider (approximately 500 µm in width) in rat aortas examined immediately after labeling. Since ECs that are experimentally induced to migrate would commence migration between 13 and 20 hours¹⁹ and then migrate at an average extrapolated rate of 16 µm/h (range 7.0 to 25.0 µm/h),^{1 2 3 4} such wounds should have been reendothelialized within 2 days after wounding and would not have significantly affected the migration of ECs toward the heart, which would resume after these wounds had been repaired.

Nonetheless, by dividing the furthest distance that the DiI-labeled ECs had moved past the proximal suture during the 3- or 6-week period by time in hours, it is possible to obtain a rough approximation of the migration rate. Given that all distance measurements were made from the suture line and that we do not know how close to that line the labeled ECs initially extended, the rates obtained would be underestimates, with the greatest values from ECs that were closest to the suture line at the time of labeling. These estimates of migration rates ranged up to 6.2 µm/h and are lower than the extrapolated rates of rat aortic EC migration (7.0 to 25.0 µm/h) in response to experimental wounds induced with either a nylon filament or an inflated balloon catheter or by freezing the intima with a cold probe,^{1 2 3 4} obtained by noting the size of the original wound and the time required to repair it.

The ECs may be migrating from regions of higher proliferation to replace ECs lost in other regions of the circulatory system. Although the normal proliferation rate of ECs of the rat aorta over a 24-hour period is extremely low (between 0.01% and 0.45%),^{20 21} there are focal areas in which the daily EC proliferation rate may be as high as 10%,²² predominantly along the ventral wall of the thoracic aorta of young adult rats²³ and along the wall of the thoracic and abdominal aorta of the pig²⁴ and the guinea pig.^{25 26} It was hypothesized that these regions may represent areas from which ECs continually migrate.²⁷ Therefore, if there are also zones of higher cell proliferation along the abdominal aorta of the rat, as there are in the thoracic aorta,²³ they could be a source of migrating ECs. However, since both the pattern²³ and distribution²⁶ of these foci of proliferating ECs varies inconsistently along the wall of the aorta, their contribution to EC migration will be difficult to evaluate.

Alternatively, it is possible that the ECs migrate primarily from regions of high cell proliferation in the capillaries. Kurz et al²⁸ showed that in the developing chick embryo there are focal zones of proliferating ECs in the capillaries and suggested that they act as a source of ECs to reestablish normal cell density in the larger vessels. Earlier, the same group also demonstrated that quail ECs will migrate centripetally within embryonic arteries.^{29 30} Therefore, if there are similar regions of high cell proliferation in capillaries of the rat, as in the chick embryo,²⁸ and if they persist throughout the life of the animal, then it is conceivable that the migration of ECs from these regions could provide a continuous source of ECs for the larger rat vessels. Interestingly, in the adult mouse, the proliferation rate of ECs in capillaries is significantly higher (0.6% to 2.4%)^{31 32 33} than the proliferation rate of ECs in the large arterial vessels of the adult rat (0.01% to 0.45%).³²

In contrast, cell loss is more prevalent at sites of high shear blood flow, commonly found at branch sites.²³ Since branches along the aorta generally increase toward the heart, one can envisage that the overall rate of cell loss would also increase in this direction. In addition, EC loss could occur within the chambers of the heart. The absence of stress fibers in most endocardial cells implies that the cells are not firmly attached to the basement membrane.³⁴ The substantial dimensional changes in ventricles from the contraction and relaxation of the heart muscle could lead to the detachment of the endocardial cells from the substratum.³⁴ Therefore, theoretically, the migration of the endothelium toward the heart from regions of cell proliferation along the aorta and the capillaries to regions of cell loss at points of high shear along the aorta and within the chambers of the heart would maintain a constant turnover of ECs.

The migrating ECs would have to adapt to a variety of environmental conditions as they move from the capillaries to the larger vessels and then along these vessels toward the heart. Such adaptations would include changes in the tight-junction complexity from discontinuous in the capillaries to continuous in the larger vessels³⁵ and in the length and thickness of stress fibers as ECs move through regions that vary in shear exerted by the blood flow.^{36 37 38 39 40 41 42} In addition to these structural adaptations, the cells would have to upregulate the production of certain growth factors and extracellular matrix proteins and downregulate others as they migrate through regions that differ in shear.^{43 44}

It would be interesting to determine whether the migration of ECs, which could play an important role in maintaining the integrity of endothelial lining, is impaired in rats older than those examined in this study or in rats that have an increased susceptibility to atherogenesis, such as the spontaneously hypertensive rat.⁴⁵

	Acknowledgments

 
This research was supported by grants from the Medical Research Council of Canada to V.I. Kalnins and N.H. McKee.

Received October 7, 1996; accepted March 14, 1997.

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