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(Circulation Research. 1997;81:311-319.)
© 1997 American Heart Association, Inc.

Articles

Flow-Induced Vascular Remodeling in the Rat Carotid Artery Diminishes With Age

Jody K. Miyashiro, Veronica Poppa, , Bradford C. Berk

From the Department of Medicine, Division of Cardiology, University of Washington, Seattle.

Correspondence to Dr Bradford C. Berk, Division of Cardiology, Box 357710, University of Washington, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu

	Abstract

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Abstract Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99±3 g) and 9 adult (199±5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by 93%, whereas flow in the contralateral carotid increased by 46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was 1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48±0.05 versus 1.29±0.04, respectively; P=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.

Key Words: outer diameter ratio • shear stress • endothelial nitric oxide synthase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular remodeling has recently been shown to play a pathophysiological role in atherosclerosis¹ and restenosis,² and the ability to remodel may influence the susceptibility to these clinical coronary artery diseases. In addition to traditional risk factors, we believe that unidentified genetic factors that influence remodeling ability will prove to be important in determining the incidence and severity of cardiovascular diseases such as atherosclerosis, hypertension, and restenosis.

It was formerly believed that the size of the plaque in atherosclerosis and restenosis was the primary determinant of vessel patency and that the vessel had only a passive static role in the stenotic process. The study of Glagov et al³ was one of the first to question this concept by demonstrating that human coronary arteries actively attempt to "accommodate" plaques by enlarging vessel diameter. Studies from several laboratories,^{2 4} including our own,⁵ have subsequently demonstrated that vascular remodeling also occurs in animal models of balloon injury and angioplasty. These studies showed that there was little correlation between neointimal mass and the extent of luminal stenosis, because the vessels had remodeled in a positive (outward) way to accommodate the obstruction⁶ or remodeled in a negative (inward) way to worsen the obstruction.

Vascular remodeling has been described in blood vessels in response to both physiological and pathological stimuli. Flow and/or pressure-induced changes in vascular structure have been well described in rabbits,^{7 8} dogs,⁹ and rats¹⁰ ; in fetal development¹¹ ; and clinically after graft placement.^{12 13 14 15} Increased pressure and wall tension are associated with a thickened vascular wall,¹⁶ whereas increased flow and shear stress are associated with enlarged luminal diameter.^{9 17 18} Shear stress and its effect on the endothelium have been proposed to be the primary mediators for flow-induced remodeling, because endothelial denudation limits the ability to remodel⁷ and because shear stress has been shown to affect the gene expression and protein function of several important mediators, including endothelial NO synthase,^{19 20} endothelium-derived relaxing factor,²¹ platelet-derived growth factor-A and -B chains,^{22 23 24} and mitogen-activated protein kinase.^{25 26} These and other yet-to-be-identified molecules are likely regulators of vascular remodeling.

Complex responses are often the result of many gene interactions, and vascular remodeling is probably no exception, given the multiple events that are activated by flow. In addition, there is a dramatic heterogeneity to the extent of plaque accommodation in atherosclerotic arteries and in the restenotic process among patients³ and no correlation with traditional cardiovascular risk factors except diabetes. The heterogeneous nature of vascular remodeling in atherosclerosis and after balloon angioplasty suggests that several genetic loci control this process. To add to the genetic complexity, the genetic predisposition for remodeling may be more strongly expressed in patients of different ages, as is the case in breast cancer.²⁷

Animal models are useful for analyzing complex genetic traits, because they permit control of environmental conditions and genetic backgrounds through selective breeding. The rat, in particular, is an ideal candidate for the study of the genetics of vascular remodeling because of its size, ease of handling, the availability of inbred strains, and the recent development of a rat linkage map.²⁸ To study the genetics of remodeling in rats, it is necessary to have a reliable quantitative method to determine the remodeling phenotype. Thus, the purpose of the present study was to develop and characterize such a rat model and to use this model to determine the effect of age on the remodeling process.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal care and experimental procedures described below were performed according to the guidelines of the National Institutes of Health and American Heart Association for the care and use of animals. All aspects of this study were approved by the University of Washington Animal Care Committee.

Development of the Model for Vascular Remodeling
To develop a rat model for vascular remodeling, we anesthetized 13 adult male outbred Sprague-Dawley rats (B&K Universal, Inc, Kent, Wash) with an intramuscular injection of ketamine hydrochloride (5 mg/100 g), xylazine (0.5 mg/100 g), and acepromazine maleate (0.5 mg/100 g). Supplemental doses of one-third the loading dose were given as needed. Arterial pressure was monitored in the femoral artery with a PE-10 catheter and Statham P23 pressure transducer. The common carotid arteries and the left internal and external carotids were exposed through a midline cervical incision.

To determine a reliable model for flow-induced vascular remodeling, blood flow in the left common carotid artery was reduced by two procedures. In one group of rats (336±52 g, n=5), the internal and external carotid arteries were ligated, and left carotid blood flow was reduced to that of the patent occipital artery (method 1). The thyroid artery was also ligated if its origin was proximal to the occipital artery. For the second group of rats (417±7 g, n=8), blood flow through the left carotid was reduced by creating a distal left common carotid artery stenosis. Silk suture (8-0 or 6-0) was tied around the left common carotid and around a piece of 3-0 monofilament suture just proximal to the carotid bifurcation (method 2). A single twist in the silk suture between the artery and the monofilament facilitated removal of the monofilament after tying.

Common carotid artery blood flows and outer diameters (ODs) were measured before the ligations and again immediately after the ligations to determine acute changes in vessel size and flow. ODs and flow were measured after flooding the area with 37°C physiological saline and after topical administration of 37°C nitroglycerin at a dose that elicited maximal vasodilation (20 µg/mL). Common carotid arterial diameters were measured with a reticule and dissecting microscope at two to three positions (2 mm apart) along the exposed vessel and averaged. Corresponding blood flows were measured with an ultrasonic transit-time volume flowmeter (Transonic Systems, Inc) while the vessels were covered with fluid (saline or nitroglycerin) to achieve acoustic contact. Zero flow on the flowmeter was checked after each placement of the flow probe by temporarily occluding the vessel with an atraumatic vascular clip. After the ligations and hemodynamic measurements were completed, the animals were allowed to recover.

The animals were reanesthetized 4 weeks later. The common carotids were reexposed, and flow, OD, and arterial pressure were measured after flooding the surgical cavity in the neck with warm saline and nitroglycerin, as described above.

We compared the effectiveness of the two methods of ligation to reduce left carotid blood flow and induce vascular remodeling. On the basis of these comparisons, we used the first method (method 1) of completely ligating the left internal and external carotid arteries to reduce left carotid blood flow in all subsequent experiments.

Remodeling in Juvenile and Adult Inbred Rats
To test whether remodeling ability varies with age, 14 juvenile (99±3-g) and 9 adult (199±5-g) male Fischer rats (Charles River Laboratories, Wilmington, Mass) were anesthetized, and their left carotid arterial branches were completely ligated using the first ligation method described above. OD and flow in the common carotid arteries were measured after flooding the surgical cavity in the neck with warm physiological saline and nitroglycerin as described above. These measurements were made before ligation, immediately after ligation, and 4 weeks after ligation. Arterial pressure was monitored via the femoral artery as described above. Eight juvenile (94±2-g) and 9 adult (211±4-g) rats underwent sham surgeries in which they were treated the same as the experimental rats (ie, the carotid arteries were isolated under anesthesia) but without ligations. All Fischer rats were maintained under pathogen-free conditions.

Dimensions of the common carotid arteries were also determined by conventional histological methods in the Fischer rats. After receiving supplemental anesthesia (pentobarbital, 0.1 mg/100 g), the rats were perfusion-fixed with 10% formalin at 110 mm Hg, a pressure that approximates systolic pressure in these animals. The vessels were dissected and immersed in formalin overnight. The tissues were then embedded in paraffin, and cross sections were stained with hematoxylin and eosin, Verhoeff–van Gieson, and orcein. Computer-assisted morphometry of the internal and external elastic laminar perimeters was performed with a digitizing board and a camera lucida attached to a standard light microscope.²⁹ OD, luminal area, and medial area were calculated from the perimeters by assuming that the vessel cross sections were circular in vivo. Four juvenile (98±2-g) and 4 adult (210±7-g) rats served as histological controls for the experimental and sham-operated rats; these animals were killed at the 0-week time point (no ligation) and perfusion-fixed in the manner described above.

The mean estimated shear stress () was calculated, assuming laminar flow, from the Hagen-Poiseuille equation as follows: =4Q/r³, where is the blood viscosity (poise), Q is the mean volume blood flow (mL/s), and r is the vessel radius (cm). Blood viscosity was assumed to be constant at 0.035 poise.¹²

To test whether small chronic flow alterations (4 to 5 mL/min differences in flow) alter endothelial NO synthase (eNOS) protein levels, we performed immunohistochemical studies on the formalin-fixed carotid arteries of ligated and sham-operated adult Fischer rats. Carotid arterial segments were cut open longitudinally, pinned luminal side up, and exposed to mouse monoclonal antibodies for eNOS (Transduction Laboratories, 1:500 dilution) and diaminobenzidine as recommended by Transduction Laboratories. We then separated the endothelium from the rest of the artery as a monolayer to facilitate visualization of the antibody complexes, as previously described.³⁰ Three independent investigators then scored the differences in eNOS expression in right and left arteries of each rat in a paired (per rat) blinded fashion. There were two possible outcomes per rat: a value of 0.1 was assigned to the situation in which the right carotid segment reacted with the eNOS antibody more than the left carotid; a value of 0 was assigned to the opposite situation. The resulting scores were summed across observers, and the sums were tested for statistical significance using Welch's t test to determine whether chronic unilateral carotid ligation affects eNOS protein concentrations.

All results are reported as mean±1 SEM. To test whether the stimulus for remodeling differed between experimental groups, independent t tests were performed on the logarithm of the percent change in flow (at 5 minutes and at 4 weeks after ligation). To test for differences in the vascular response to chronic flow alterations, independent t tests were performed on the logarithm of the percent change in ODs (for each side) and on the logarithm of the ratio of right to left ODs (ODRs). Differences in luminal area, medial area, estimated ODs, and log(estimated ODR) were also compared using independent t tests. Multiple comparisons were possible using the Bonferroni correction. All statistical tests were done with SYSTAT for the MacIntosh, version 5.2.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Carotid vascular structure changed after 4 weeks of chronic flow alterations. Generally, vessels became smaller with decreased flow (left carotid) and became larger with increased flow (right carotid) (Fig 1). Maximal vasodilation with nitroglycerin (topical, 20 µg/mL) did not eliminate the contrast between right and left carotids that developed in response to flow.

View larger version (52K): [in this window] [in a new window]   Figure 1. Cross sections of the common carotid arteries 4 weeks after distal left carotid branch ligation. A, Left (flow-reduced) carotid. B. Right (flow-augmented) carotid. Orcein-stained sections show that the carotid diameter and luminal area are larger in the flow-augmented artery than in the reduced-flow artery. IEL indicates internal elastic lamina; EEL, external elastic lamina. Bar=0.2 mm.

Development of the Model
To develop a quantitative flow-dependent remodeling technique, we compared two techniques of flow reduction in outbred adult Sprague-Dawley rats that were based on previous work. Analogous to the model of Langille and colleagues,^{7 8} we performed complete ligation of the left internal and external carotid arteries (method 1). Analogous to the model of Guyton and Hartley,¹⁰ we performed a partial distal ligation of the left common carotid artery using a combination of monofilament and silk sutures (method 2). The second method has the theoretical advantage of decreasing flow to an extent that is dependent on the gauge of the monofilament spacer and independent of relative vessel anatomy. However, this method proved to be impractical because of the technical difficulty in keeping the monofilament suture parallel to the vessel while tying the silk suture perpendicular to the vessel axis. Furthermore, complete ligation of left carotid branches (method 1) caused a greater and more reproducible decrease in left common carotid blood flow than did partial distal ligation of the left common carotid (method 2) (with a decrease from 7.3 to 0.5 mL/min [a 92.9% decrease] versus 5.9 to 1.5 mL/min [a 74.6% decrease], respectively) (Table). The ratio of right to left ODs after 4 weeks, which we have termed the ODR, was correspondingly greater in rats with completely ligated branches (1.53±0.07) than in rats with partial ligation (1.13±0.10). The ODR was unaffected by 20 µg/mL nitroglycerin (1.50±0.09 in rats with complete ligation and 1.15±0.09 in the partially ligated rats). Therefore, complete ligation of the left carotid branches resulted in a more reproducible and greater flow reduction than did distal stenosis and, consequently, greater vascular remodeling. On the basis of these characteristics, complete ligation appears to be a better model for vascular remodeling and was used for all subsequent experiments.

View this table: [in this window] [in a new window]   Table 1. Comparison of Two Methods of Reducing Left Carotid Blood Flow

Remodeling in Juvenile and Adult Inbred Rats: Change in Vessel Flow
To determine the effect of age on vessel remodeling, we compared juvenile (99±3-g) and adult (199±5-g) rats. At the end of the 4-week experimental period, ligated juvenile rats weighed 216±4 g, juvenile sham-operated rats weighed 212±5 g, ligated adult rats weighed 267±4 g, and adult sham-operated rats weighed 276±6 g.

In juvenile and adult Fischer rats, complete ligation of the left distal carotid branches decreased left common carotid blood flow (to 8.4±1.2% and 6.4±1.0% of initial flow, respectively; P=.445). These decreases were still apparent 4 weeks after ligation (13.4±2.4% and 22.5±2.1% of initial flow in juveniles and adults, respectively; P=.219) (Fig 2). Flow on the contralateral side correspondingly increased immediately after ligation of the left carotid branches (by 49±12% in juvenile rats and 43±10% in adult rats, P=.831) and remained elevated 4 weeks after ligation (resulting in a 121±27% increase from initial flow values in juvenile rats and a 148±55% increase in adult rats, P=1.00).

View larger version (15K): [in this window] [in a new window]   Figure 2. Common carotid artery blood flow before and 4 weeks after ligation of the left carotid branches. A, Juvenile rats. B, Adult rats. In juvenile rats, left carotid flow decreased from 2.27±0.15 to 0.21±0.04 mL/min after ligation. In adult rats, left carotid flow decreased from 2.63±0.14 to 0.16±0.02 mL/min after ligation. Blood flow immediately increased in the right carotid of both groups. Blood flow remained altered for 4 weeks. Juvenile and adult rats were exposed to equivalent flow alterations (the percent change in flow did not differ significantly between juvenile and adult rats). indicates initial right carotid flow; , right carotid flow at 4 weeks; , initial left carotid flow; and , left carotid flow at 4 weeks.

There were no significant differences between right and left carotid flows (initially or after 4 weeks) in either sham-operated juvenile or sham-operated adult animals. In sham-operated rats, flow increased in both carotids over the 4-week experimental period. In sham-operated juvenile rats, flow increased from 2.80±0.17 to 3.92±0.36 mL/min in the right carotid and from 3.05±0.16 to 4.28±0.27 mL/min in the left carotid. In sham-operated adult rats, flow increased from 4.04±0.25 to 4.44±0.21 mL/min in the right carotid and from 4.37±0.21 to 4.95±0.30 mL/min in the left carotid during the experimental period.

Remodeling in Juvenile and Adult Inbred Rats: Change in Vessel Dimensions
The ODs of juvenile rat carotids changed to a significantly greater extent than did the ODs of adult rat carotids after 4 weeks (Fig 3) despite similar changes in flow. Specifically, juvenile rats tended to show a greater response to increased flow than did adult rats: OD increased by 28.5±4.6% in juvenile rats compared with 10.8±4.8% in adult rats (P=.058). However, there was no difference between juvenile and adult rats in the response to decreased flow: OD decreased by 15.5±3.5% in juvenile rats and by 16.4±3.4% in adult rats (P=1.000). Arterial pressures were comparable between these groups at the 4-week time point (96±1 mm Hg in juvenile rats and 102±2 mm Hg in adult rats).

View larger version (15K): [in this window] [in a new window]   Figure 3. Common carotid artery outer diameter (OD) (in vivo) before and 4 weeks after ligation of the left carotid branches. A, Juvenile rats. B, Adult rats. There was a smaller percentage increase in size of adult right carotids compared with juvenile carotids (P=.058) despite the same increase in right carotid flow (see Fig 2). The responses of juvenile and adult rats to reduced flow were not significantly different. indicates initial right carotid OD; , right carotid OD after 4 weeks; , initial left carotid OD; and , left carotid OD after 4 weeks.

Rats that underwent a sham operation were used to evaluate normal vascular growth. In juvenile rats that underwent sham procedures, OD increased from 0.79±0.02 to 0.91±0.02 mm in the right carotid and from 0.81±0.01 to 0.93±0.03 mm in the left carotid (data not shown). These values, which represent 15.4±2.6% and 14.6±2.7% increases in the right and left ODs, respectively, tended to be different from the changes observed in experimental juvenile rats during increased flow (P=.087) and during decreased flow (P<.001). The absolute OD of each carotid also differed from that of experimentally ligated juvenile rats (right, P=.002; left, P<.001).

In sham-operated adult rats, OD increased from 0.99±0.02 to 1.10±0.02 mm on the right and from 0.98±0.02 to 1.05±0.02 mm on the left, representing 12.1±2.1% and 7.0±2.4% increases, respectively. When analyzed as absolute ODs or as percent changes in OD, these vascular changes were significantly different from adult vessels that were exposed to decreased flow (P.001) but not different from those exposed to augmented flow (P=.284). The observed difference between the ODs of experimental animals and those of the age-matched sham-operated animals is due to flow-induced vascular remodeling. Compared with adult rats, juvenile rats exhibited a greater remodeling response to increased blood flow. This contrast was not attributable to differences between juvenile and adult growth rates, because sham-operated juvenile and adult rats showed the same degree of vascular growth over the 4-week experimental period.

The effect of topical nitroglycerin (20 µg/mL) on carotid OD was nearly undetectable, although there was a modest effect on flow (flow in both carotids of all groups increased by 6% to 12%, before as well as 4 weeks after ligation). This is expected, since the vessel radius does not have to increase significantly to cause a profound increase in flow (resistance to flow is proportional to 1/radius⁴ ). Thus, maximal vasodilation with nitroglycerin did not affect the changes in vessel dimension that had occurred with the chronic alterations in flow, indicating that these changes were primarily structural and not attributable to altered vascular tone.

Eight ligated juvenile rats, one ligated adult rat, and one sham-operated juvenile rat were excluded from histological analysis because of differences in tissue-processing methodology. Morphometry of formalin-fixed paraffin-embedded specimens confirmed the differences between vessels that were observed by reticle in vivo, although the histological differences were less striking. In experimental juvenile rats, the right and left ODs were 0.74±0.01 and 0.62±0.01 mm, respectively, compared with 0.67±0.01 and 0.69±0.01 mm in sham-operated juvenile rats. In experimental adult rats, the right and left diameters were 0.74±0.01 and 0.67±0.01 mm, respectively, compared with 0.75±0.02 and 0.73±0.01 mm in adult sham-operated rats. There was a smaller contrast between right and left OD of experimental animals compared with that observed in vivo. This difference is primarily due to the fact that the carotid diameters determined from histological cross sections were 20% to 25% smaller than diameters determined in vivo by reticule because of the exclusion of adventitia in the histological measurements and tissue shrinkage during fixation.³¹ In addition, adventitia may not be distributed proportionately among vessels, so its exclusion may decrease the difference between the right and left ODs. Nonetheless, the OD results from histological determinations were qualitatively similar to the in vivo reticule measurements: juvenile right and left ODs were significantly affected by unilateral ligation compared with the sham operation (right, P<.001; left, P<.001), whereas in adult rats, only left ODs were affected by unilateral ligation compared with sham operation (right, P=1.000; left, P<.001).

Chronic flow alterations influenced not only OD but luminal area as well. In juvenile and adult rats, luminal area significantly decreased in the left (reduced flow) carotid compared with the left carotid in sham-operated rats (Fig 4, upper panel). However, luminal area increased in the right (increased flow) carotid only in juvenile rats. Luminal area significantly decreased in the left (reduced flow) carotid compared with the left carotid in sham-operated rats and increased in the right (increased flow) carotid of juvenile but not adult rats (Fig 4, upper panel). In contrast, the medial area was significantly altered only in the right carotid of juvenile ligated rats compared with sham-operated rats (P=.023; Fig 4, lower panel). Geometric analysis of these findings indicates that the media must be thicker in the flow-reduced carotids than in the flow-augmented carotids.

View larger version (34K): [in this window] [in a new window]   Figure 4. Juvenile and adult cross-sectional areas. Top, Luminal areas. Bottom, Medial areas. Luminal and medial areas generally tended to increase slightly over the 4-week time course of the experiment. The juvenile carotid luminal area was more responsive to chronic flow alterations compared with the adult carotid luminal area. Only the right carotid medial area was significantly affected by flow alterations. Control indicates the 0-week time point; sham, sham-operated rats; Exp, experimentally ligated rats; solid bars, left common carotid artery; and open bars, right common carotid artery.

We postulated that there should be a causal relationship between changes in vessel flow and vessel diameter on the basis of previous investigations.^{7 8 10} Consistent with this hypothesis, we found that there was a direct monotonic relationship between flow and diameter: decreases in the flow reduced OD in all rats, and increases in flow enlarged OD. A difference in the sensitivity to flow (of borderline significance) was observed between juvenile and adult carotids that were exposed to increased flow (Fig 5): there appeared to be greater remodeling in juvenile than in adult rats.

View larger version (14K): [in this window] [in a new window]   Figure 5. Relationship between flow and outer diameter (OD) (in vivo) before and 4 weeks after ligation. A, Juvenile rats. B, Adult rats. OD decreased in response to reduced flow in both groups. In contrast, the vessels of juvenile rats showed greater enlargement with increased flow than did those of adult rats. Thus, the slope of the flow-OD relationship tended to be steeper in juvenile compared with adult rats. indicates right carotid at 0 weeks; , right carotid at 4 weeks; , left carotid at 0 weeks; and , left carotid at 4 weeks. Arrows show the direction of change.

Remodeling in Juvenile and Adult Inbred Rats: Change in Vessel Shear Stress
Endothelial cells have been proposed to be the primary mediators of vessel remodeling⁸ by their ability to sense and respond to changes in shear stress. Wall shear stress, which is related directly to flow and inversely to diameter, is maintained within a narrow range (10 to 30 dyne/cm²) under normal physiological conditions. Presumably, this narrow range for physiological shear stress is a consequence of dynamic control of OD in response to flow to maintain constant shear stress.⁹ Accordingly, we found that shear stress tended to decrease in the sham-operated animals over the 4-week experimental period despite moderately increased flow (values are in legend of Fig 6).

View larger version (15K): [in this window] [in a new window]   Figure 6. Flow and wall shear stress before ligation, immediately after ligation, and 4 weeks after ligation. A, Juvenile rats. B, Adult rats. Immediately after ligation of the distal left carotid branches, shear stress and flow decreased in the left () and increased in the right () carotid. Wall shear stress stayed lower in the left carotid of both groups after 4 weeks of altered flow. However, in the flow-augmented right carotid, juvenile and adult rats responded differently. In juvenile rats, OD increased sufficiently after 4 weeks to restore wall shear stress to initial values, whereas wall shear stress remained elevated in the adult rats. indicates right carotid at 0 weeks; , right carotid at 4 weeks; , left carotid at 0 weeks; and , left carotid at 4 weeks. Arrows show the direction of change. In sham-operated animals, shear stress was maintained over the 4-week time course (the average right and left carotid shear stress was 35±1 dyne/cm2 initially and 30±2 dyne/cm2 after 4 weeks in juvenile animals; in adult animals, the average shear stress was 28±2 dyne/cm2 initially and 27±3 dyne/cm2 after 4 weeks).

After distal left carotid ligation, shear stress acutely decreased in the left carotid and increased in the right carotid (Fig 6). In juvenile rats, shear stress in the flow-augmented right carotid artery recovered to initial values after 4 weeks (from 29.2±3.6 to 29.4±3.5 dyne/cm²) (Fig 6) because of an increase in OD. In adults, however, structural changes in the flow-augmented artery were inadequate to restore shear stress to initial values, and shear stress increased from 16.8±1.6 to 27.8±2.6 dyne/cm². These results indicate that a chronic increase in flow causes a greater increase in OD and smaller change in shear stress in juvenile compared with adult rats.

In response to decreased flow (left carotid), we found that shear stress decreased and never recovered in either group. Shear stress decreased by 73.2±4.2% in juvenile rats and by 60.5±4.5% in adult rats after 4 weeks of reduced flow.

The ODR as a Measure of Vascular Remodeling
The age-related difference in remodeling can be summarized by a comparison of the ODRs at 4 weeks (Fig 7). Initially, the right and left ODs are equal in each rat, and the ODR is 1.0. However, juvenile and adult rats differ significantly after left carotid ligation. The juvenile ODR becomes 1.48±0.05 after 4 weeks, reflecting a high degree of remodeling; in contrast, the adult ODR is only 1.29±0.04 (P=.030). The ODR computed from histological morphometry also showed that juvenile rats experience more extensive remodeling than do adult rats (ODR, 1.23±0.03 versus 1.11±0.03, respectively; P=.025).

View larger version (29K): [in this window] [in a new window]   Figure 7. The outer diameter ratio (the ratio of right to left outer diameter) represents the ability to remodel. Initially, the outer diameter ratio was 1.0 in juvenile and adult rats. After 4 weeks, the outer diameter ratio in juvenile rats increased more than in adult rats (P=.030).

eNOS Protein Levels Are Sensitive to Small Chronic Changes in Flow
Rats that had undergone distal left carotid ligation showed higher eNOS expression in the right (flow-augmented) carotid arterial segments than in the left (flow-reduced) segments (Fig 8). There was no difference in eNOS expression between sham-operated right and left carotid arteries. This sidedness of eNOS expression differed significantly between ligated and sham-operated animals (P=.025).

View larger version (50K): [in this window] [in a new window]   Figure 8. Chronic carotid flow alterations affect endothelial NO synthase (eNOS) protein concentrations. Immunohistochemistry of the en face endothelial layer shows more immunoreactive eNOS in the increased flow artery (right carotid, B) compared with the reduced flow artery (left carotid, A). Bar=25 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of the present study are that flow-induced vascular remodeling is age dependent and correlates with maintenance of constant shear stress and increased expression of eNOS relative to the reduced flow vessel. In a highly reproducible rat model of chronic (4-week) carotid flow alteration, we found that juvenile Fischer rats experience more successful vascular remodeling than do adult Fischer rats, as measured by changes in OD, luminal area, and shear stress. Young rats maintained a constant wall shear stress despite chronic increases in flow, whereas adults failed to normalize shear stress. In addition, 4 weeks of left carotid flow reduction and right carotid flow augmentation resulted in an ODR that was significantly greater in juvenile rats than in adult rats. The procedures developed and characterized in the present study represent an excellent model to study and quantify vascular remodeling in response to alterations in flow.

The finding that young rats experience more successful vascular remodeling than do adult rats confirms our hypothesis that age is an important factor that influences the extent of remodeling in rats. Our findings are in general agreement with the findings of Langille and colleagues,^{8 18} in which young rabbit carotid arteries exhibited a greater decrease in diameter in response to decreased flow⁸ and a greater increase in diameter in response to increased flow¹⁸ than did adult rabbit carotid arteries. Unlike Langille and colleagues, we found no age-related effect on the response to reduced flow, but this difference may be due to species or to the extent of flow reduction.

One of the primary goals of the present study was to develop and characterize a simple, highly reproducible model of blood flow reduction in order to study and quantify flow-induced vascular remodeling in rats. Guyton and Hartley¹⁰ used a silver clip to restrict rat carotid flow by 35% (compared with the unclipped side after 5 to 10 weeks). The resulting ODR was only 1.11, a value that is not very different from what one would expect to find in the nonligated animal (Fig 7). Langille and colleagues^{7 8} ligated the left external carotid artery of rabbits and decreased flow by 70%. The resulting ODR was 1.5. Although this is a reliable way of decreasing flow in rabbits, ligation of the left external carotid alone does not result in a large flow decrement in rats (J.K. Miyashiro, unpublished data, 1995). Our method of ligating both the left internal and external carotid arteries in rats consistently decreased the ipsilateral common carotid flow to a greater extent than was reported in the studies described above (left carotid flow decreased to 8.4±1.2% of initial flow in juvenile rats and to 6.4±1.0% of initial flow in adult rats) while increasing contralateral flow by 48.9±1.2% and 42.8±9.8%, respectively. The resulting ODRs were significantly different from those in sham-operated animals (Fig 7). Importantly, right and left carotids were exposed to the same pressures in each animal because the ligations were distal to the areas of interest. Our method (by virtue of its reproducibility, the large flow changes compared with previous studies, and the consequently large effects on vascular dimensions) provides us with a tool for quantifying and comparing remodeling between rat strains, ages, and treatments.

The reproducibility and accuracy of in vivo reticule measurements were validated in the present study. In vivo measurements of carotid OD by reticule correlated well with measurements from histological cross sections. The histological measurements were generally smaller than the in vivo measurements, although both methods attempted to measure systolic dimensions. The larger OD obtained from in vivo measurements is due to the inclusion of adventitia, but tissue shrinkage during fixation could also contribute to the difference in measurements.³¹ Both methods show that juvenile rats experience significantly more remodeling than do adult rats, but the in vivo method will be a useful way to detect changes in vessel dimensions in response to flow without having to kill the animal.

Medial cross-sectional area increased in juvenile rats with increased flow but did not change in adult rats. Increased flow did not affect medial thickness in either group. These effects on medial mass and thickness in juvenile rats are in agreement with the findings of Brownlee and Langille¹⁸ in young rabbits. In reduced-flow arteries, however, the medial area did not decrease with luminal area and OD, indicating that the media is thicker in the reduced-flow arteries compared with increased-flow arteries and arteries from age-matched sham-operated animals. These results are in contrast to previous studies in which the medial area decreased with flow reduction and thickness was unchanged.^{8 10} The extent of ligation in the present model may explain the differences in response; it is possible that extreme flow reductions invoke additional or alternative remodeling mechanisms. The present results suggest that existing vascular components migrated and rearranged themselves to achieve a change in luminal area and/or that a balance of cell proliferation and cell death in the vessel wall was maintained as part of the remodeling process. Future studies are needed to confirm these ideas.

Endothelial cells are the primary sensors of wall shear stress and are thought to play an important role in the remodeling process through the release of products such as NO and growth factors.²⁴ Our data emphasize the importance of the endothelium in the remodeling process in two ways: (1) we show a correlation between changes in shear stress and remodeling, and (2) we show a flow-dependent difference in eNOS expression. In the present study, we demonstrated that shear stress acutely increased with augmented flow, but after 4 weeks, the OD increased and restored shear stress toward initial values, especially in the juvenile rats. In the ligated carotid, shear stress acutely decreased and then partially recovered toward initial values when the OD decreased after 4 weeks. Juvenile and adult rats were exposed to similar acute changes in shear stress, but the resulting effect on vascular structure was greater in juvenile rats. Thus, the ability to remodel in response to shear stress appeared to decline with age.

Our finding that unilateral carotid ligation results in a right versus left carotid difference in eNOS expression that is not present in sham-operated animals confirms that small chronic changes in flow (differences of 4 to 5 mL/min or a shear stress difference of 25 dyne/cm²) can modulate eNOS levels in vivo. Although an effect of flow on eNOS mRNA, protein, and NO production has previously been demonstrated in vivo for large chronic flow alterations (increases of >100 mL/min, as occurs in the aortic fistula model),³² to our knowledge this is the first study to demonstrate such an effect in vivo with small chronic flow alterations. These data support a role for eNOS in vascular remodeling. This hypothesis is strengthened by the work of Tronc et al,¹⁷ who demonstrated that the eNOS inhibitor N^G-nitro-L-arginine methyl ester decreases the extent of remodeling in rabbits with carotid fistulas.

Physiological remodeling may also occur in response to wall tension. Increased shear stress is associated with increased luminal area, whereas increased wall tension is associated with increased wall thickness. Wall tension was not an important stimulus for remodeling in the present model, because each artery (ie, within each rat) was exposed to the same pressure. It is conceivable, however, that wall tension may begin to play a role in vascular structure once flow-induced changes have occurred: wall tension decreased in the reduced-flow arteries compared with sham-operated control arteries because of the decrease in vessel radius and increase in wall thickness but was not affected by chronic increases in flow.

Differences in whole-body growth rates cannot fully explain the age-related differences in carotid artery remodeling. The sham-operated animals represent normal vessel growth and development. When the experimental animals are paired with their sham-operated controls, the difference in the carotid OD at the 4-week time point is due to flow-induced remodeling rather than growth. Even with this normalization, juvenile animals exhibit greater remodeling than do adult animals, although the final flow-augmented vascular dimensions are about the same. Presumably, the final dimensions (OD, flow, and shear stress) are about equal because head size and heart weight do not change proportionate to body weight.³³ Blood flow increases in sham-operated animals but not as much as OD and body weight. Thus, shear stress is generally high in young rats but decreases slightly with normal vessel development. Therefore, the optimal time to study remodeling is in the juvenile animal, when the potential for remodeling is highest.

The decrement in remodeling ability with age may be due to changes in endothelial and smooth muscle cell function or amounts of structural components. Endothelial regulation of vascular tone reportedly declines with age.^{34 35} This decline may be due to a reduced ability to sense or transduce the shear stress stimulus, resulting in altered production of NO or other vasoactive products.³⁶ Alternatively, smooth muscle cell reactivity to vasoactive products may decrease.³⁷ Finally, age-related changes in the matrix of vessel walls³⁸ may also affect remodeling potential.

The present study demonstrates that age affects flow-induced vascular remodeling. Therefore, future studies evaluating genetic determinants of remodeling or treatment effects should be performed in juvenile animals before the response of interest has declined. Other factors, such as environment, may also affect the expression of remodeling genes. An important aspect of the present study was that experiments were carried out with inbred Fischer rats under controlled pathogen-free conditions, which likely reduced experimental variability. A useful index of remodeling ability established in this rat model is the ratio of the right and left common carotid OD (termed ODR). Determination of ODR does not require animals to be killed and therefore allows breeding of animals based on remodeling ability for future genetic analysis.

	Acknowledgments

 
This work was supported by National Institutes of Health grants R01 HL-49192 to Dr Berk and T32 HL-07312 to Dr Miyashiro. Dr Berk is an Established Investigator of the American Heart Association. The authors would like to thank Yu Xiang for surgical assistance and for help in performing morphological measurements.

	Footnotes

 
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received October 3, 1996; accepted June 6, 1997.

	References

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