Shear Stress Is Differentially Regulated Among Inbred Rat Strains
An important compensatory response to atherosclerosis is vascular remodeling, with maintenance of vessel lumen diameter and shear stress. Both hemodynamic and environmental factors contribute to vascular remodeling and shear stress regulation, and the process is probably also influenced by genetic factors. To establish an animal model for genetic analysis of shear stress regulation and vascular remodeling, we studied the effects of chronic flow alteration in four inbred rat strains. By ligating the left internal and external carotid arteries, we caused a ≈90% decrease in left common carotid blood flow and a ≈50% increase in right (contralateral) common carotid flow. After 4 weeks of altered flow, there were significant interstrain differences with respect to the change in carotid outer diameter (OD), the relationship between flow and shear stress, and the extent to which shear stress was normalized. Genetically hypertensive rats (GH) exhibited the greatest reduction in shear stress in response to increased flow, stroke-prone spontaneously hypertensive rats (SHR-SP) exhibited a smaller response, and Brown Norway (BN) rats exhibited the smallest response. SHR-SP and GH also differed significantly in outward remodeling (defined as an increase in lumen and vessel diameter) in increased flow arteries. In response to decreased flow, BN rats exhibited the smallest reduction in shear stress. These findings demonstrate significant strain-dependent differences in shear stress regulation and vascular remodeling in response to altered flow. This study emphasizes the important role of genetic factors in vascular remodeling and suggests that genetic analysis of these strains will provide novel insights into the underlying mechanisms.
Treatment of chronic vascular disease has largely focused on reducing intimal mass to maintain adequate lumen size.1 However, Glagov2 demonstrated in 1987 that intimal mass is not the primary factor that determines lumen size in human coronary arteries because vessels remodel to accommodate increasing plaque burden. More recently, intravascular ultrasound (IVUS) has confirmed that vessels can either enlarge (remodel outward) or constrict (remodel inward) at sites of atherosclerosis and after balloon angioplasty.3,4 These observations suggest that vascular remodeling is an important determinant of vessel lumen size and may alter the clinical course of atherosclerosis.
Despite the clinical significance of this process, the mechanisms responsible for vascular remodeling are largely unknown. An important stimulus for vascular remodeling is likely to be blood flow. As blood flows along a vessel, it creates shear stress on the vessel wall. It has been well established that the development of atherosclerosis is related to shear stress with an increased predilection for atherosclerosis to develop in regions of low shear stress and/or turbulence.5 Endothelial cells may act as the sensors and mediators of this hemodynamic regulation of atherogenesis by virtue of their ability to produce antiatherogenic factors such as nitric oxide (NO). Shear stress and endothelial cells may also play a role in flow-induced remodeling,6 in an effort to maintain physiological levels of shear stress (10 to 20 dyn/cm2). NO appears particularly important, based on studies with inhibitors of endothelial nitric oxide synthase (eNOS)7 and eNOS knockout mice.8
Flow-induced remodeling occurs rapidly3 and is age-dependent.3 Beyond this, however, our knowledge of the remodeling process is limited. In the clinical setting, the degree of vascular accommodation to plaque formation appears to be continuous and varies greatly among individuals,2,4 a feature that is characteristic of a polygenic process. It is further suggestive of a differential sensitivity to blood flow and shear stress.
The mechanisms responsible for vascular remodeling and regulation of shear stress are likely to be complex and polygenic; therefore, efforts to understand the genetic basis for these processes are better approached in animal models such as the mouse and rat. We studied chronic flow alterations in rat carotid arteries to identify and characterize strains that exhibit significantly different responses. Because genetic analysis of physiological phenomena requires contrasting phenotypes, the goal of the present study was to determine whether inbred rat strains exhibit polymorphic phenotypes, such as the magnitude of inward and outward remodeling, and the ability to maintain physiological levels of shear stress.
Materials and Methods
Rat Strains, Surgical Procedures, and Hemodynamic Measurements
Animal care and experimental procedures were performed according to National Institutes of Health and American Heart Association guidelines for the care and use of animals. All aspects of this study were approved by the University of Rochester and University of Washington Animal Care Committees.
The following inbred rat strains were used in this study: BN (Harlan Sprague-Dawley, Indianapolis, Ind), Fischer 344 (Fischer; Charles River, Wilmington, Mass), GH (University of Otago, Otago, New Zealand), and SHR-SP (Oregon Primate Research Center, Beaverton, Ore). Juvenile male rats (80 to 120 g) of each strain underwent left carotid artery (LCA) ligation as described previously, resulting in left common carotid flow reduction by ≈90% and increased right carotid artery (RCA) flow by ≈50% in rats.3
Before and immediately after ligation, we measured the common carotid OD using a dissecting scope and eyepiece reticule. Common carotid blood flow was measured using an ultrasonic transit-time flowmeter (Transonic Systems Inc, Ithaca, NY). These measurements were repeated after 4 weeks of remodeling. All flow and OD measurements were also measured after a topical application of nitroglycerin (NTG, 20 μg/mL) to the exposed carotids. Sham-operated rats (ie, surgery without ligations) served as controls. ΔOD (OD at 4 weeks−OD baseline) and Δshear stress (shear stress at 4 weeks−shear stress baseline), or the % ΔOD (ΔOD/OD baseline×100) and % Δshear stress (Δshear stress/shear stress baseline×100) were chosen as the parameters for comparison between strains, to account for differences at baseline. Thus, substantial outward remodeling in response to increased flow is associated with a high ΔOD, and substantial inward remodeling in response to decreased flow is associated with a low ΔOD. Changes in shear stress (or ΔOD) after 4 weeks of chronic flow alteration were also normalized to their respective shams by subtracting the Δshear stresssham (or ΔODsham) from Δshear stressligated (or ΔODligated). Normalization controlled for the effect of growth on vessel dimensions. Anesthetized arterial blood pressure was also measured via the femoral artery at baseline and at the 4-week time point.
The mean estimated shear stress (τ) was calculated, assuming laminar flow, from the Hagen-Poiseuille equation as τ=4ηQ/πr,3 where η is the blood viscosity (poise), Q is the mean volume blood flow (mL/sec), and r is the vessel radius (cm). Blood viscosity was assumed to be constant at 0.035 poise.
Perfusion Fixation of GH and SHR-SP Carotid Arteries
Rats were perfusion-fixed 4 weeks after ligation or sham-operation with 10% formalin at mean arterial pressures, as described.3 Arteries were divided into 3 divisions: upper, middle, and lower. The upper and lower segments were paraffin embedded, and 4-μm-thick cross sections were cut and stained with hematoxylin and eosin. Morphometric analysis was performed with MCID software (Imaging Research, Inc) by a blinded observer.3 Four cross sections per vessel were used for analyses, and the average was used to determine the group average.
Immunohistochemistry of Carotid Arteries
Tissues were processed for immunohistochemistry as previously described.9 In brief, BrDU tablets were placed subcutaneously in rats under anesthesia, 24 hours before euthanasia. The primary anti-BrDU antibody (DAKO) was applied (1:40) and visualized with a biotinylated secondary antibody (Vector Laboratories) using diaminobenzidine as the chromagen. BrDU-positive cells were determined manually as the number of positive cells per media cross-sectional area by a blinded observer. Sections of ileal tissue were processed as positive controls for each animal. For en face immunohistochemistry, we used eNOS (1:500; Transduction Labs) and CD31 (PECAM; 1:500; gift from Dr Keigi Fujiwara, Center for Cardiovascular Research, University of Rochester, Rochester, NY) visualized with rhodamine conjugated goat antimouse (1:1000) and fluorescein conjugated goat anti-rabbit (1:1000) antibodies, respectively.
Results are reported as the mean±1 SEM. The major determinants of Δshear stress were evaluated using stepwise regression analysis. To test for interstrain differences in Δshear stress, ΔOD, absolute OD, body weight, heart weight, or blood pressure, analysis of variance followed by a Fisher’s post hoc test was performed. All statistical tests were done with Statview for Macintosh, version 5.0.1.
We anticipated that the carotid ligation model would cause ≈90% reduction in flow in the LCA and ≈50% increase in flow in the RCA based on our previous study.3 To verify that the flow stimulus was the same for all strains, we measured flow in the carotid arteries before and after LCA ligation. Initial flow in the common carotid arteries varied between 1.8 to 3.2 mL/min. Ligation of the left internal and external carotid arteries immediately decreased left common carotid flow to ≈0.2 mL/min and increased right common carotid flow to ≈2.5 to 4.8 mL/min. The percent changes in flow did not differ significantly between strains (Figure 1). To determine the relative importance of rat strain, change in blood flow, OD, body weight, and mean arterial pressure (MAP) in determining change in vessel shear stress, we performed stepwise regression analyses (online Table 1, available in the online data supplement at http://www.circresaha.org). These analyses identified that the Δshear stress (week 4−week 0) in both RCA and LCA was significantly determined by strain, Δblood flow, ΔOD, and Δbody weight (P<0.0001). Although body and heart weights varied significantly between strains (online Table 2), simple regression analysis did not indicate significant relationships with Δshear stress or ΔOD, suggesting that differences in growth rate were not a major factor in the remodeling response. Similarly, although there were MAP differences among strains after 4 weeks of ligation (online Table 2), regression analyses showed that MAP was not a significant determining factor.
The mechanisms for remodeling in response to decreased flow (LCA) are likely to differ from remodeling in response to increased flow (RCA); therefore, the results for each carotid are described separately.
Right Carotid Arteries
Measurements of OD and flow used to calculate shear stress were made before and 4 weeks after ligation as described (Table, top). To determine the contribution of vascular tone to ΔOD and Δshear stress, we repeated measurements after topical application of NTG (Table, bottom). Regulation of shear stress in the RCA was significantly different in the 4 rat strains studied. Previously, we found that Fischer rats maintained a constant shear stress of ≈30 dyne/cm2 in the RCA 4 weeks after ligation of the LCA.3 In the present study, GH, Fischer, and SHR-SP maintained shear stress at the baseline physiological level; however, shear stress increased significantly (+36%) in the RCA of BN rats (Figure 2A). ΔShear stress in SHR-SP carotids 4 weeks after ligation compared with GH (Figure 2A) was statistically significant (P=0.018).
Because strain-dependent characteristics other than change in flow may also influence remodeling, we studied sham-operated animals for each rat strain as controls. The Δshear stress in the sham groups exhibited interstrain variation, likely due to differences in blood flow and OD over the 4-week experimental period (see online Table 3). To control for these strain-dependent differences in hemodynamic parameters, we normalized the Δshear stress in the carotid artery of ligated animals to the Δshear stress in sham-operated animals by subtraction, (% Δshear stressligated−% Δshear stresssham; Figure 2B). The results for the normalized shear stress calculation showed changes in shear stress in the order, GH<Fischer<SHR-SP∼BN. Although the ordering of strains remained similar to the order shown in Figure 2A, it is apparent that the greatest effect of normalization occurred in SHR-SP arteries. Hence, shear stress was maintained at physiological levels in GH and Fischer rats in response to increased flow, whereas shear stress increased significantly in SHR-SP and BN rats.
We also evaluated the role of vascular tone in the Δshear stress by local application of NTG to fully relax the carotids (Figure 2C and Table, bottom). We determined the percent Δshear stress in the presence of NTG [calculated as (shear stress at 4 weeks NTG−shear stress at baseline NTG)/shear stress at baseline NTG×100)]. We found percent Δshear stress after NTG application changed in the order, GH<Fischer<SHR-SP<BN, identical to the data in Figure 2A. These results indicate that the differences in shear stress among strains are primarily due to changes in structure rather than changes in vascular tone.
Because OD was shown to be a major determinant in shear stress regulation by stepwise regression analyses (online Table 1), we examined the ΔOD in the various strains. There were significant differences between strains (Figure 3A) in the order GH>Fischer>SHR-SP>BN, precisely the converse of the trend observed for Δshear stress. We also examined the ΔODs normalized to their respective shams and found that strain differences were smaller after normalization, although the order remained GH>Fischer>SHR-SP∼BN (not shown). After application of NTG to remove vasoconstrictor tone, there was only a small increase in OD in the RCAs of all strains (Figure 3B), indicating that the ΔODs were mainly a reflection of altered structure (compare Figures 3B and 3A).
Because strain was a major factor in shear stress determining shear stress, we focused further analyses on GH and SHR-SP strains, two hypertensive strains that displayed contrasting abilities to regulate shear stress. It is noteworthy that blood flow after ligation increased acutely by a similar magnitude in GH and SHR-SP (≈49% and ≈48%, respectively). To gain insight into the mechanisms for these differences we performed two comparisons: (1) shear stress against flow and (2) OD against flow (Figure 4).
To determine whether there was a difference in shear stress and flow relationships between GH and SHR-SP strains, we plotted Δshear stress against Δflow (Figure 4A, top). We found that the regression slope for GH RCAs was +1.7 (not significantly different from zero), which is consistent with appropriate regulation of shear stress over a 5-fold variation in flow (1 to 5 mL/min). Compensatory regulation of shear stress would be indicated by a line with slope equal to zero. The finding that most points are less than zero is a consequence of shear stress in GH RCAs actually decreasing over the 4-week experimental period. In contrast, the slope for SHR-SP carotids was +4.9 (significantly different from zero), and reflects the inability to maintain normal shear stress with increasing flow. The relationship between shear stress and flow after application of NTG (Figure 4A, bottom) did not change from that observed in control conditions for either SHR-SP or GH rats. This suggests that the differences in regulation between GH and SHR-SP were structural in origin, rather than dependent on vasoconstrictor tone.
We next examined the relationship between Δflow and ΔOD (Figure 4B). This analysis indicated that GH RCAs exhibit a larger increase in OD for a given change in flow than SHR-SP carotids. In fact, the slope for SHR-SP was ≈0, suggesting an impaired sensitivity to flow (Figure 4B, upper). We determined the extent to which these differences were due to vasoconstrictor tone by application of NTG and found that there was no significant effect of NTG on GH or SHR-SP arteries (Figure 4B, bottom), again indicating a vascular structural alteration.
To further characterize the differences in the structural response to flow, we determined alterations in vessel wall compartment areas by histomorphometry (Figure 5). GH and SHR-SP RCAs after 4 weeks of ligation exhibited increases in lumen cross-sectional area of 18% and 6%, respectively, compared with sham-operated vessels. In contrast, there were decreases in media cross-sectional area of 4% and 13% in GH and SHR-SP RCAs, respectively, compared with control vessels. The adventitia cross-sectional area also demonstrated divergent strain effects with a 6% decrease in GH, and a 13% increase in SHR-SP carotids. No intima formation was observed in any RCA.
Left Carotid Arteries
In response to decreased flow in the LCAs, all strains with the exception of BN showed large decreases in shear stress of ≈75% (Figure 2A). Normalization of the Δshear stress in the LCAs of the ligated animals to the respective shams did not significantly change the results for the individual strains (Figure 2B). After application of NTG, there was also no significant difference in the results for Δshear stress (compare Figures 2C and 2A). This indicates that the strain differences in LCA shear stress were not due to differences in vascular tone.
We determined the percent ΔOD among the strains and found that OD decreased in the order GH<SHR-SP∼Fischer∼BN (Figure 3A). Normalization of ΔOD to shams resulted in a similar order, GH<SHR-SP∼BN∼Fischer (not shown). There was no significant change in the OD measured in the presence of NTG in any strain (compare Figures 3B and 3A).
Because the RCAs of GH and SHR-SP strains displayed significant differences in the relationships of flow with shear stress and OD, we also examined these relationships in the low-flow LCAs (Figure 6). The plot of Δshear stress versus Δflow (Figure 6A, top) showed a similar relationship under control conditions in GH and SHR-SP LCAs in contrast to the difference observed for the RCAs. The relationship between shear stress and flow was not affected by application of NTG in either strain (Figure 6A, bottom). We next determined the relationship between Δflow and ΔOD in LCA arteries. GH and SHR-SP vessels exhibited similar decreases in OD for a given decrease in blood flow (Figure 6B, top). There was no effect of NTG on this relationship in either strain (Figure 6B, bottom).
Histomorphometry showed that after 4 weeks of ligation, the lumen area was reduced by 16% and 23% in the LCA arteries of GH and SHR-SP rats, respectively, relative to corresponding vessels from sham-operated rats (Figure 5). The media and adventitia cross-sectional areas exhibited small changes of 2% to 8% in both strains that were not significantly different from their respective shams. There was no intima formation in any LCA.
Immunohistochemical Analyses of Medial Cell Proliferation and eNOS Expression
To evaluate possible mechanisms that may account for strain-dependent differences in remodeling, we characterized medial cell proliferation and eNOS expression, processes that previously have been suggested to play roles in vascular remodeling. Cell proliferation as measured by media BrDU incorporation at 4 weeks was higher in GH rats under basal conditions compared with SHR-SP (online Table 4). In the RCAs of ligated groups, we observed 51% and 29% decreases in GH and SHR-SP carotid media cell proliferation, respectively. Cell proliferation in LCAs was also reduced to a similar extent in response to ligation (54% and 15% in GH and SHR-SP, respectively). There were no gross changes in the number of elastic lamina or their integrity assessed by elastin staining with van Gieson stain.
Endothelial integrity was demonstrated by en face CD31 staining, which showed an intact and morphologically normal endothelium that did not differ among strains or among sham versus ligated (online Figure 1, available in the online data supplement at http://www.circresaha.org). Expression of eNOS at weeks 0 and 4 demonstrated significant differences comparing strains and sham versus ligated (Figure 7). eNOS expression was significantly greater in GH than SHR-SP in shams (≈2-fold, Figure 7f versus e), and in GH than SHR-SP for both ligated LCA and RCA (Figure 7a versus c, 7b versus d). Importantly the ligated RCA showed increased total eNOS (≈4-fold) in GH compared with SHR-SP.
The major findings of this study are as follows: flow-induced vascular remodeling (assessed by changes in shear stress and vessel OD) differs significantly in four inbred rat strains; strain-specific responses to increased and decreased blood flow differ; and comparison of two genetically hypertensive strains showed that GH rats are more sensitive to increases in blood flow (measured by the relationship between flow and both OD and shear stress) than SHR-SP. We believe that the differences in vascular remodeling and shear stress in these highly inbred strains reflect important effects of strain-specific gene expression. These data suggest that a genetic cross among the inbred strains would yield phenotypic differences that could be used to identify key regulatory genes.
In an earlier study in our laboratory, we demonstrated that juvenile Fischer rats remodeled to a greater extent than adult Fischer rats in response to chronic flow alterations, and showed that shear stress is a key physiological force that correlates with the extent of vascular remodeling.3 Based on these findings, we used juvenile rats to compare remodeling phenotypes between strains. The present study extends our earlier study in Fischer rats by demonstrating that there are major strain-dependent differences in the regulation of shear stress. A possible limitation of the present model is the complexity involved in using anesthesia to permit measurements of blood flow and ligation surgery, 4 weeks of observation, and repeat hemodynamic analysis under anesthesia. It is noteworthy, however, that our surgical success rate was 100%. The potential usefulness of this model could be confounded by growth effects on gene expression, but we think this is unlikely because body weight was not a significant factor in our stepwise regression analysis. Moreover, it is likely that fundamental mechanisms of remodeling are similar in both juvenile and adult animals.
The mechanisms for regulation of vessel diameter and shear stress in response to alterations in flow remain to be defined but likely include important genetic determinants. This concept is supported by our findings in the GH strain where shear stress was maintained at normal physiological levels at all values of flow (a “flat” slope of shear stress versus flow). In the SHR-SP strain, however, shear stress increased with increasing flow (a “steep” slope of shear stress versus flow). Shear stress is related directly to flow and inversely to OD as described by Poiseuille’s Law. In accordance with this, we identified the major determinants of shear stress regulation to be change in flow and OD by stepwise regression analyses. Because the magnitude of the flow stimulus was similar between strains, there are several potential explanations for differences in shear stress regulation. First, there may be differences in the expression or activity of flow-sensing mechanisms such as integrin-matrix interactions, caveolae, G proteins, or ion channels. Second, there may be differences in flow transduction mechanisms as a consequence of differential expression and/or coupling of molecular signaling pathways such as NO-dependent events (see below). Finally, there may be differences in the integrated cellular response to vascular remodeling such as differences in migration and proliferation, matrix production, and turnover.
Although the underlying processes in vascular remodeling are poorly understood, flow-dependent outward remodeling has been shown to involve eNOS and NO. Both mice deficient in eNOS8 and rabbits infused with the eNOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME) fail to remodel appropriately.7 These studies suggest that NO derived from eNOS may be critical for outward remodeling. It should be noted that the role for NO may be vascular bed–specific because L-NAME has been shown not to restrict outward remodeling in mesenteric arteries.10 The results of the present study suggest appropriate endothelial production of NO in GH rats because they exhibited compensatory outward remodeling and shear stress regulation. We have recently found ≈80% vasorelaxation by acetylcholine in phenylephrine-constricted GH rat aortae in vitro (Ibrahim J, unpublished data, 2002), indicating appropriate endothelial function in this strain. This is further supported by a study indicating that the l-arginine/NO system is not deficient in GH rats.11 The greater eNOS expression in GH carotid arteries compared with SHR-SP at baseline fits well with this report and with our functional data for acetylcholine vasorelaxation. Based on the greater eNOS expression in GH carotid arteries relative to SHR-SP both in sham and ligated animals, we speculate that differences in NO may explain the greater outward remodeling observed in the GH. Our data with NTG suggest that the function of NO is not to increase vasorelaxation, but more likely reflects changes in gene expression mediated by the transcriptional effects of NO. In addition, there may be endothelial dysfunction in SHR-SP as reported by other investigators.12–15 Specifically, vascular production of reactive oxygen species, including superoxide (which consumes NO to form peroxynitrite, ONOO−), has recently been shown to be greater in SHR-SP than in Wistar-Kyoto rats.15 Other proteins that may potentially influence remodeling include matrix metalloproteinases and their inhibitors,16 cytokines such as TGFβ,17,18 growth factors such as PDGF,19 cytoskeletal proteins such as vimentin,20 and proteins associated with apoptosis or cell proliferation.21,22 The exact role of these candidate proteins in vascular remodeling is currently under study by a number of laboratories.
Our results for cell proliferation and vessel compartment areas provide insight into potential mechanisms for differences in remodeling, especially between GH and SHR-SP. Surprising findings were that cell proliferation and media area decreased in the RCA, despite the fact that the RCA exhibited increases in lumen and vessel area. Most revealing was the finding that media area in the SHR-SP decreased to a greater extent than GH, almost equivalent to the differences in lumen area in GH and SHR-SP. These findings suggest a potential negative correlation between decreases in media area and increases in lumen area. The decrease in cell proliferation and media area observed in the RCA may be due to increased smooth muscle cell apoptosis. Apoptosis has been previously observed with vascular remodeling in response to both injury and flow.21,23 Based on these findings, it is possible that SHR-SP carotid smooth muscle cells are more sensitive to proapoptotic signals than GH carotid smooth muscle cells.
In summary, this study shows that there is a strong genetic basis for shear stress regulation and vascular remodeling. By using inbred populations with distinct phenotypic flow responses, future studies using global expression techniques (eg, genetic crosses to identify quantitative trait loci, cDNA microarrays, and proteomics) may elucidate the genetic basis of remodeling.
This work was supported by NIH grant HL-62826 to B.C.B. and an AHA Scientist Development Grant to J.M.K. We thank David Nagel and Mary Georger for help with histomorphometry and immunohistochemistry.
- Received August 16, 2002.
- Revision received March 21, 2003.
- Accepted March 21, 2003.
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