Vascular Medicine. Atherosclerotic Lesion Size and Vulnerability Are Determined by Patterns of Fluid Shear Stress

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1 Vascular Medicine Atherosclerotic Lesion Size and Vulnerability Are Determined by Patterns of Fluid Shear Stress Caroline Cheng, PhD; Dennie Tempel, BSc; Rien van Haperen, BSc; Arjen van der Baan, BSc; Frank Grosveld, PhD; Mat J.A.P. Daemen, MD, PhD; Rob Krams, MD, PhD; Rini de Crom, PhD Background Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies. Methods and Results We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, versus ); contained fewer smooth muscle cells ( % versus %), less collagen ( % versus %), and more lipids ( % versus %); and showed more outward vascular remodeling (214 19% versus 117 9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II induced intraplaque hemorrhages occurred in the lowered shear stress regions only. Conclusions Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions. (Circulation. 2006;113: ) Key Words: atherosclerosis hemodynamics inflammation mechanical stress plaque The occurrence of atherosclerosis is closely linked to local hemodynamic factors. Shear stress, the drag force acting on the endothelium as a result of blood flow, is thought to play a critical role in the development of endothelial dysfunction and atherosclerosis. This concept is based on the observation that atherosclerotic plaque formation occurs preferentially in areas such as the inner curvatures of coronary arteries, where shear stress is low ( 1.5 N/m 2 in humans), or near bifurcations, where shear stress is oscillatory (ie, displaying directional change and boundary layer separation). 1 3 Conversely, straight arterial segments with laminar flow in humans have shear stress levels of 1.5 N/m 2 and appear to be protected from atherosclerosis. The relation between shear stress and atherosclerosis is based almost exclusively on observational studies in humans and large animals. 1,4,5 In addition, there is a large body of evidence on the response of cultured endothelial cells to variations in shear stress (reviewed in Shyy and Chien 6 ). Editorial p 2679 Clinical Perspective p 2753 Although this previous work provides insight into the relationship between shear stress and plaque development, direct proof that deviations in shear stress induce atherosclerosis is lacking. To provide such evidence, an appropriate in vivo model that can generate complex shear stress fields is required. Furthermore, it is unclear whether low shear stress and vortices with oscillatory shear stress have different atherogenic properties. Therefore, investigators in our laboratory have recently developed a perivascular shear stress modifier (referred to as a cast) that can induce changes in shear stress patterns in vivo in a straight vessel and in a defined manner. Placement of the cast creates lowered shear stress upstream from the cast, increased shear stress in the cast, and oscillatory (ie, bidirectional, with vortices) shear stress downstream from the cast (Figure 1). 7 In the present study, we used this model to assess the effect of in vivo alterations of shear stress on the Received September 27, 2005; revision received March 16, 2006; accepted April 4, From the Departments of Cardiology, Thoraxcenter (C.C., D.T., A.v.d.B., R.K.), and of Cell Biology and Genetics (R.v.H., F.G., R.d.C.), Erasmus MC, University Medical Center, Rotterdam; the Department of Pathology (M.J.A.P.D.), Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht; and the Department of Vascular Surgery (R.d.C.), Erasmus MC, University Medical Center, Rotterdam, The Netherlands. The online-only Data Supplement can be found at Correspondence to Rini de Crom, PhD, Department of Cell Biology and Genetics, Erasmus MC, Room Ee1073, Dr Molewaterplein 50, 3015 GD Rotterdam, The Netherlands. m.decrom@erasmusmc.nl 2006 American Heart Association, Inc. Circulation is available at DOI: /CIRCULATIONAHA

2 Cheng et al Shear Stress and Atherosclerosis 2745 Figure 1. Schematic representation of the shear stress patterns induced by the cast. On the right is a straight segment of a mouse carotid artery without a cast, which has laminar blood flow (indicated by parallel arrows). Based on Doppler measurements, the average shear stress has been calculated to be 15 N/m 2. On the left a mouse carotid artery with the cast is shown. Upstream from the cast, shear stress is relatively low (compared with the shear stress in the control vessel), 10 N/m 2, caused by the flow-limiting stenosis induced by the cast. Within the cast, shear stress increases from relatively low ( 10 N/m 2 ) to relatively high ( 25 N/m 2 ) because of the tapered shape of the cast. Downstream from the cast, shear stress is oscillatory ( 10 N/m 2 ; range of 60 N/m 2 ). development of atherosclerosis in apolipoprotein E deficient (apoe / ) mice. Our findings reveal that atherosclerotic lesions develop under conditions of both lowered shear stress and vortices with oscillatory shear stress within 6 weeks of cast placement, whereas no lesions develop in the increased shear stress region. In addition, we analyzed the composition of these lesions. We demonstrate that lowered shear stress induces the development of extensive lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce the growth of stable lesions. Using angiotensin II administration, we show that intraplaque hemorrhages occur exclusively in the atherosclerotic lesions of lowered shear stress regions. Methods An expanded Methods section is available in the online-only Data Supplement. Animals All experiments were performed in compliance with institutional (Erasmus MC, Rotterdam, The Netherlands) and national guidelines. In Vivo Alteration of Shear Stress To induce standardized changes in shear stress, we used a shear stress modifier (referred to as a cast) that is identical to the device we described previously (Figure 1; see also the online-only Data Supplement). 8 The cast imposes a fixed geometry on the vessel wall and thereby causes a gradual stenosis, resulting in increased shear stress in the vessel segment inside the cast, a decrease in blood flow and consequently a lowered shear stress region upstream from the cast, and a vortex downstream from the cast (oscillatory blood flow). The upstream reduction in blood flow is caused by a device-induced, flow-limiting stenosis of 70%. The downstream vortex is generated by a boundary separation immediately downstream from the cast, which is induced by a combination of velocity acceleration at the beginning of the cast, inertia of the blood, and the angle of the streamline at the end of the cast. Control casts consisted of a similar cylinder made of the same material with a continuous, nonconstrictive diameter. The shear stress values have been derived from measurements of vessel diameter and the velocity of blood flow (Doppler measurements). 8 The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written. Results Lowered Shear Stress and Oscillatory Shear Stress Both Induce Atherosclerotic Plaque Formation, Whereas Increased Shear Stress Protects Against Atherosclerosis First, the actual shear stress levels were calculated in both undisturbed and cast-instrumented carotid arteries in mice (Figure 1). These appeared to be much higher than the shear stress values commonly reported in humans. However, the cast induced variations in shear stress as expected: a 30% decrease in shear stress upstream from the cast (lowered shear stress), increased shear stress inside the cast, and vortices with oscillatory shear stress downstream from the cast. To study the effect of the 3 different shear stress fields on lesion formation, apoe / mice were fed an atherogenic diet, instrumented with the cast, and humanely killed at different times (6, 9, and 12 weeks). Whole-mount lipid staining of aortic arches and carotid arteries showed that atherosclerotic lesions had already developed at the earliest time (Figure 2A) in the inner curve of the aortic arch and at the beginning of the side branches. These are natural sites of shear stress related plaque initiation, 9 as we confirmed in mice without casts (Figure IA in the online-only Data Supplement). In addition, lipid deposits had started to develop in the lowered shear stress and the vortices/oscillatory shear stress regions in the carotid artery instrumented with the cast at 6 weeks. These were found along the length of the carotid artery, where shear stress is relatively low (Figure IB in the online-only Data Supplement). By 9 and 12 weeks, the lesion areas had grown markedly larger in the lowered shear stress regions (Figure 2B and 2C). In the vortices/oscillatory shear stress regions, a gradual increase in lesion area was also evident (Figure 2A through 2C), but these were considerably smaller than the lesions in the lowered shear stress regions. No atherosclerotic lesions

3 2746 Circulation June 13, 2006 Figure 2. Lowered shear stress and vortices with oscillatory shear stress induce atherosclerosis in apoe / mice fed an atherogenic Western diet. Whole-mount aortic arches and carotid arteries were stained with Oil red O for atherosclerotic lesions. White lines demarcate the previous position of the cast with the increased shear stress region. Upstream from the cast is the lowered shear stress region, and downstream from the cast are the vortices with an oscillatory shear stress region. Animals were instrumented with a cast 2 weeks after starting the diet and were humanely killed after 6 (A), 9 (B), or 12 (C E) weeks of cast placement. No lesions were detected in the carotid arteries of either sham-operated mice (D) or animals instrumented with a nonconstrictive cast (E). Representative images are shown from groups consisting of 6 to 8 animals. were present in the increased shear stress region at the earliest time, and moderately sized lesions developed only in 2 of 7 mice (29%) at 9 weeks and in 3 of 6 mice (50%) at 12 weeks after cast implantation, whereas all animals developed lesions under lowered and oscillatory shear stress from week 6 onward. At the latest times, atherosclerotic lesions extended into the most upstream part of the vessel segment within the cast. Controls included the contralateral, nontreated carotid arteries, sham-operated animals (Figure 2D), and animals treated with a nonconstrictive cast (Figure 2E). No lipid staining was observed under any of these conditions, not even after 12 weeks of cast placement. Plasma cholesterol levels were increased in animals fed a Western diet (at 6 weeks, mmol/l [ mg/dl]; at 9 weeks, mmol/l [ mg/dl]; and at 12 weeks, mmol/l [ mg/dl]) compared with animals fed a normal chow diet ( mmol/l [ mg/dl]). Plasma cholesterol concentrations between animals treated with the cast and the nonconstrictive cast were not different (at 12 weeks with the cast, mmol/l [ mg/dl] versus at 12 weeks with the nonconstrictive cast, mmol/l [ mg/dl]). These results indicate that the diet, surgical procedure, or the material of the cast did not contribute to the observed atherosclerotic lesion formation in the straight segment of the carotid artery. Lowered Shear Stress Is Associated With More Extensive Lesions Than Oscillatory Shear Stress Figure 3A contains photomicrographs displaying carotid artery morphology after 9 weeks of cast placement in the lowered, increased, and vortices/oscillatory shear stress regions, as well as in the contralateral carotid artery (undisturbed shear stress). In the increased shear stress region, the appearance of the vessel is very similar to that of the undisturbed, control region. In contrast, in the lowered and oscillatory shear regions, atherosclerotic lesion formation is obvious. However, the lesions have a strikingly different morphology. Platelet endothelial cell adhesion molecule-1 staining revealed an intact endothelium in all regions studied at all times (shown for the 9-week point in Figure 3B). Measurements of intima-media ratio showed that the atherosclerotic lesion areas in the lowered shear stress regions were much more extensive than those in the regions with vortices and oscillatory shear stress ( versus , respectively; Figure 3C). In both regions, plaque size was significantly larger than in the undisturbed region. In contrast, no significant difference in intima-media ratio was observed between the increased shear stress region and the undisturbed region (Figure 3C). Vascular remodeling was evaluated by comparing the cross-sectional area of the vessel wall obtained from the lowered and the oscillatory shear stress regions of the instrumented carotid artery and the untreated contralateral carotid artery (control region). Remodeling was observed in the lowered shear stress region, where the relative cross-sectional vessel area was 2-fold higher compared with the control region, whereas there was no significant change in the region with vortices and oscillatory shear stress (Figure 3D). At 6 and 12 weeks, similar differences in intima-media ratios and in vascular remodeling between the different shear stress regions were found (data not shown).

4 Cheng et al Shear Stress and Atherosclerosis 2747 Figure 3. Histological analyses of carotid arteries 9 weeks after cast placement in apoe / mice fed a Western diet. A, Representative images are shown from regions with lowered (low), increased (high), and vortices with oscillatory (oscill.) shear stress, as well as from the contralateral, nontreated carotid artery with undisturbed (undist.) shear stress (hematoxylin and eosin staining; original magnification, 10). B, Representative images show platelet endothelial cell adhesion molecule-1 staining (red) of intact endothelium in the 4 different shear stress regions. Autofluorescence of the elastic lamellae is green, and the cell nuclei stained with DAPI are blue. Original magnification, 100. Histological analyses were performed by quantification of the intima-media ratio (C) and of the relative cross-sectional area of the vessel wall (D), which was obtained by measuring the area confined by the external elastic lamina of the instrumented carotid artery and expressed as a percentage of the cross-sectional area of the contralateral, nontreated carotid artery. *P 0.05 vs lowered shear stress. P 0.05 vs vortices/oscillatory shear stress. Lowered Shear Stress Induces Lesions With a Vulnerable Plaque Phenotype, Whereas Vortices With Oscillatory Shear Stress Induce Development of Stable Lesions To examine the effect of lowered shear stress and vortices with oscillatory shear stress on plaque composition, we performed (immuno)histochemical analyses 9 weeks after cast placement. Because no lesions developed in control vessels, the percentage of plaque components in the intimal area of these vessels could not be analyzed and are therefore excluded. Macrophages and lipids were abundantly present in lesions of both lowered and oscillatory shear stress regions (Figure 4A). Quantification of macrophage-positive areas showed no relative difference between lesions in the lowered shear stress and oscillatory shear stress regions (Figure 4B). The lipid content, however, was significantly higher in the lesions located in the lowered shear stress regions than in those in the oscillatory shear stress regions ( % versus %, respectively; Figure 4B). Further analysis of lesion components revealed that lowered shear stress lesions contained only thin layers of smooth muscle cells and collagen in the cap of the lesion, whereas in the oscillatory shear stress lesion, smooth muscle cells and collagen-positive areas were more uniformly distributed in the intima (Figure 4A). This pattern was also observed at the other times studied (data not shown). The lowered shear stress lesions contained fewer vascular smooth muscle cells ( % versus %) and less collagen ( % versus %) than the oscillatory shear stress lesions at week 9 (Figure 4B). To assess the observed reduction in collagen, matrix metalloproteinase (MMP) activity was measured by DQ gelatinase assay (Figure 4A). An increase of approximately one third in MMP activity was found in the lowered shear stress region compared with the oscillatory shear stress region (Figure 4B). Expression of Proatherogenic Inflammatory Mediators Is More Prominent in the Lowered Shear Stress Region Gene expression analysis revealed increased expression of proinflammatory mediators in the lowered shear stress region compared with that in the region with vortices and oscillatory shear stress (Figure 5). Expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1, involved in the adhesive interactions between endothelial cells and leukocytes, was upregulated by 3-fold in the lowered shear stress region compared with controls, whereas only VCAM-1 was 50% upregulated in the oscillatory shear stress region. Expression of C-reactive protein was increased by 3-fold in the lowered shear stress region and by 2-fold in the oscillatory shear stress region compared with controls. Expression levels of proatherogenic vascular endothelial growth factor were increased by 5-fold in the lowered shear stress lesions exclusively. Finally, expression of the proinflammatory cytokine interleukin 6 (IL-6) was increased by 14-fold in the lowered shear stress and by 2-fold in the oscillatory shear stress region compared with controls. Intraplaque Hemorrhages Are Exclusively Observed in Lesions of the Lowered Shear Stress Region To obtain further evidence of plaque instability in the lowered shear stress region, a larger group of mice was

5 2748 Circulation June 13, 2006 Figure 4. A, Cross sections of carotid arteries taken 9 weeks after cast placement from apoe / mice fed a Western diet ( 100 magnification) and stained for macrophages, 34 lipids (oil red O), vascular smooth muscle cells (anti -actin), or collagen (picrosirius red) in the lowered shear stress (low sh. str.) and the vortices/oscillatory shear stress (oscill. sh. str.) region. MMP activity was studied by DQ gelatinase assay. B, Quantification of macrophages, lipids, vascular smooth muscle cells, and collagen in the intimal area of the lowered shear stress (low) and the vortices/oscillatory (oscill.) shear stress region and of MMP activity. Data are the mean values of sections from 5 different animals. VSMC indicates vascular smooth muscle cell. *P 0.05 vs lowered shear stress. studied after 9 weeks of cast placement. Intraplaque hemorrhages, an established sign of plaque vulnerability, was observed in 4 of 14 animals (28%). Traces of blood plasma and (partly degraded) erythrocytes were found in the basal region of the plaque close to the internal elastic lamina or in the necrotic core, whereas plaque integrity was preserved (Table and Figure 6A and 6B). Intraplaque hemorrhages were never observed in lesions of the regions with vortices and oscillatory shear stress. We tested whether an increase in blood pressure would produce an even more pronounced vulnerable plaque phenotype. 10,11 To this end, angiotensin II was chronically administered to the mice via osmotic minipumps (400 ng kg 1 min 1 ) during the last 2 weeks of a 9-week period of cast placement. The efficacy of the treatment was validated by an increase in mean blood pressure (from 95 3 to mm Hg; P 0.001). More extensive intraplaque hemorrhages were observed in atherosclerotic lesions in the lowered shear stress region at an increased frequency (6 of 8 animals [75%] compared with the control group; Table and Figure 6C, 6E, and 6F). Angiotensin II stimulation did not induce intraplaque hemorrhages in the lesions initiated by vortices with oscillatory shear stress (Figure 6D). Deposition of iron, the product of degraded hemoglobin and suggestive of intramural thrombus, was detected by the Prussian blue reaction (Figure 6F). In several cases, atherosclerotic lesions became detached from the vascular wall, causing a false lumen (Figure 6G and 6H). Plaque rupture or erosion was not detected, even after angiotensin II stimulation. Discussion In this study, we provide evidence that variations in shear stress patterns profoundly affect the initiation of atherosclerosis and induce the development of atherosclerotic plaques with a vulnerable phenotype. We conclude that both lowered shear stress and vortices with oscillatory shear stress are highly proatherogenic because (1) well-developed lesions were present from the earliest time point (week 6) in all of the cast-treated animals under both hemodynamic conditions; (2) lesions were absent in the straight segments of the contralateral, nontreated carotid arteries; and (3) the area of increased shear stress in the carotid arteries treated with the cast was protected from atherosclerotic lesion formation. In addition, lowered shear stress is a key factor in induction of plaques with a vulnerable phenotype. It should be noted that lowered shear stress means relatively low, compared with the average shear stress in a straight vessel segment, like the nontreated, contralateral carotid artery. The reason that shear stress is lowered in the vessel upstream from the cast is that the flow rate is decreased as a result of the stenosis induced by the cast.

6 Cheng et al Shear Stress and Atherosclerosis 2749 Figure 5. Vascular gene expression levels of proinflammatory mediators involved in the progression of atherosclerosis in carotid arteries 9 weeks after cast placement in apoe / mice fed a Western diet. Shown are the effects of lowered shear stress (low) versus vortices with oscillatory shear stress (oscill.) on the expression of VCAM-1, intercellular adhesion molecule-1, C-reactive protein, vascular endothelial growth factor, and IL-6 measured by quantitative polymerase chain reaction. Data were obtained from a pool of 10 animals from 4 different polymerase chain reactions. ICAM-1 indicates intercellular adhesion molecule-1; CRP indicates C-reactive protein; VEGF indicates vascular endothelial growth factor. *P 0.05 vs lowered shear stress. The actual shear stress in the lowered shear stress area is in fact high when compared with absolute shear stress values in humans. The fact that the absolute level of average shear stress is much higher in mice than in humans does not necessarily mean that shear stress cannot be studied in mice. For instance, the same is true for heart rate, as cardiac function has been extensively studied in (genetically modified) mouse models. However, it is critical to know whether the same predilection sites for atherosclerosis, which are attributed to local shear stress patterns, occur in mice and humans. We found that in apoe / mice that were fed the same Western diet as used in the present study, but without cast treatment, atherosclerosis was present in the inner curvature of the aortic arch and at the beginning of the side branches (Figure IA in the online-only Data Supplement). This pattern suggests that (relatively) low shear stress (present in the inner curvature) and oscillatory shear stress (expected at the beginning of the side branches) are involved in the development of atherosclerosis. Thus, the pathobiological consequences remain the same, even if the absolute values of shear stress are different in different species. To the best of our knowledge, mean shear stress values have not been accurately described in mice. At present, this is being studied in more detail in our laboratory (authors unpublished observations). Based on measurements in rabbits, 8 it is presumed that also in mice there are spatiotemporal oscillations in shear stress downstream from the cast. We cannot measure this directly in mice, however, because of limitations in resolution. Still, we believe that flow reversal does occur downstream from the cast in the mouse model, because in that area, we observed changes in cell shapes in mice transgenic for endothelial nitric oxide synthase (enos) fused to green fluorescent protein. 8 To obtain additional evidence for the occurrence of vortices, we performed a detailed analysis using computational fluid dynamics. We reasoned that to document the occurrence of vortices, we needed to focus on changes in direction of the velocity vectors and not on changes in amplitude. Therefore, we have displayed the normalized, spatially averaged velocity vectors on the vessel wall (Figures II and III in the online-only Data Supplement). A sudden change in direction was noted in the numerical calculations immediately downstream from the cast. Because this location coincides with the expected location of the vortex, we believe that this is another indication of a vortex in our model. Damage to the endothelium due to cast placement also could have elicited a nonspecific atherogenic response and reduced shear stress responsiveness. However, platelet endothelial cell adhesion molecule-1 staining disclosed an intact endothelium in all shear stress regions examined after 9 weeks of cast placement (Figure 3B). Furthermore, Distribution of Cast-Induced Atherosclerotic Lesions Showing the Occurrence of Intraplaque Hemorrhages With and Without Angiotensin II Stimulation Shear Stress Region Control (n 14) Angiotensin II (n 8) Lowered Vortices/Oscillatory Lowered Vortices/Oscillatory Intraplaque hemorrhages False lumen % Intimal area * * 0 Percentages are expressed as mean SEM of the area in the plaque occupied by bleeding in the animals with intraplaque hemorrhages. *P 0.05 vs vortices with oscillatory shear stress. P 0.05 vs control group.

7 2750 Circulation June 13, 2006 in a previous study, we showed that the endothelium is continuous immediately after cast placement. 8 No atherosclerotic lesions were present in the increased shear stress regions 6 weeks after cast placement. These findings are in agreement with the generally accepted notion that plaques do not develop under relatively high shear stress conditions Upregulation of enos expression could be part of the antiatherogenic properties of increased shear stress. 15 Indeed, we previously found that enos is elevated in cast-induced increased shear stress vessel segments. 8 At later times, only small lesions were found in some of the treated apoe / mice (lesion size was not different from that in the control region). At later times, atherosclerotic lesions will extend from the lowered shear stress region (upstream from the cast) into the most Figure 6. Intraplaque hemorrhages in lesions from the lowered shear stress region. ApoE / mice were fed a Western diet and instrumented with a cast around the carotid artery. A and B, After 9 weeks, intraplaque hemorrhages were found in 28% of the animals analyzed. Arrowheads point to accumulations of erythrocytes near the internal elastic lamina (A) or in the necrotic core (B). C H, After 7 weeks of cast placement, the animals were treated with an angiotensin II infusion (400 ng kg 1 min 1 ) delivered via a subcutaneously implanted osmotic minipump (Alzet model 2004; Durect Corp, Maastricht, The Netherlands) and humanely killed 2 weeks later. Intraplaque hemorrhages were frequently observed (in 6 of 8 animals). C, Overview of an atherosclerotic lesion with intraplaque hemorrhages from a lowered shear stress region. D, Overview of an atherosclerotic lesion in the vortices/oscillatory shear stress region. No intraplaque hemorrhages were detected. E, Detail of an intraplaque hemorrhage in a lowered shear stress atherosclerotic lesions. F, Same detail as in E, stained for iron deposits (arrowheads) with the Prussian blue reaction. G, Overview of a carotid artery with a large false lumen (FL). H, Detail showing the depressed lumen (arrowheads). Seven-micron cryosections were stained with eosin and hematoxylin. Representative photomicrographs from 6 animals are shown. Original magnifications are 100 (C, D, G) and 400 (A, B, F, G, H). upstream part of the vessel within the cast. Although shear stress in this vessel segment is already higher than that in the vessel segment immediately upstream from the cast, it is probably still below the average shear stress levels found in the contralateral carotid artery. The highest levels of shear stress are found in the most downstream part of the cast, which invariably remained free from lesions. Placement of a nonconstrictive sham cast did not induce atherosclerosis in the upstream and downstream regions. Thus, the cast material or design per se did not cause a nonspecific inflammatory response in the vessel wall that might have led to atherosclerotic disease. It has been reported that placement of a perivascular, nonconstrictive cuff can induce intimal hyperplasia or atherosclerosis in the treated vessel area. 16 This is remarkable, because in our

8 Cheng et al Shear Stress and Atherosclerosis 2751 control experiments, placement of the nonconstrictive sham casts did not induce such a response. Development of atherosclerosis in those cuff models could have been elicited by either the material or the surgical procedure itself. More important, the cuffs were placed around the femoral arteries, which could have responded differently to placement of the device than the carotid arteries used in this study. The superficial location of the femoral artery and movement of the hindlimbs by the animal could have limited blood flow in cuffed vessels for periods of time, resulting in lowered shear stress and the induction of atherosclerosis. Although the relation between shear stress and atherogenesis has been well documented, to the best of our knowledge, nothing is known about the effect of this hemodynamic force on plaque composition. Vulnerable atherosclerotic lesions are characterized by high percentages of destabilizing components (lipids and macrophages) and low percentages of stabilizing components (vascular smooth muscle cells and collagen). Morphologically, the destabilizing components accumulate in pools underneath a thin, fibrous cap, with little infiltration of vascular smooth muscle cells. 17,18 Shear stress could determine the vulnerability of the lesion by altering the gene expression of endothelial cells (eg, upregulation of adhesion molecules, proinflammatory factors, and factors that mediate vascular wall permeability) and by increasing the interaction of proatherogenic components in the blood (lipoproteins and monocytes) with the activated endothelium We found that lowered shear stress lesions contained more lipids, fewer vascular smooth muscle cells, and less collagen compared with lesions in the oscillatory shear stress region. In addition, these lesions had thin, fibrous caps containing few vascular smooth muscle cells. Conversely, in the lesions induced by vortices with oscillatory shear stress, a thick, fibrous cap heavily infiltrated with vascular smooth muscle cells covered the lesion, which contained similar relative amounts of macrophages but fewer lipids than did the lesions in the lowered shear stress regions. Furthermore, outward vascular remodeling, which is considered one of the characteristic features of vulnerable plaques, 22 was more prominent in the lowered shear stress region. These findings indicate that oscillatory shear stress induces the growth of more stabilized plaques, whereas lowered shear stress initiates the development of lesions with a vulnerable phenotype. This finding was further substantiated by the occurrence of intraplaque hemorrhages (both before and after administration of angiotensin II) in the lesions located in the lowered shear stress region. Intraplaque hemorrhages are considered prominent markers of plaque instability. 11,18,23,24 Without angiotensin II stimulation, the more extensive lesions that developed in the lowered shear stress regions showed small intraplaque hemorrhages close to the internal elastic lamina in 28% of the treated animals. Hemorrhages were never observed in the lesions in oscillatory shear stress regions. Angiotensin II administration increases mechanical strain on the lesions by raising blood pressure. In addition, it has been shown that angiotensin II infusion into hyperlipidemic mice augments lesion formation independently of elevations in blood pressure by eliciting a Th1 response 25 or by increasing the angiogenic properties of the plaque. 26 Despite the multipotent ability of angiotensin II to affect plaque vulnerability, our data show that intraplaque hemorrhages occurred in the lowered shear stress lesions only in response to angiotensin II stimulation. To further investigate the mechanism by which shear stress determines lesion vulnerability, we hypothesized that different shear stress patterns elicit divergent proatherogenic inflammatory responses in the vascular wall. VCAM-1 and intercellular adhesion molecule-1 expression is known to be shear stress responsive in cultured endothelial cells We confirmed those findings in vivo and found that lowered shear stress induced higher expression levels than did vortices with oscillatory shear stress, which might result in elevated leukocyte recruitment. Although there was no difference in the relative macrophage content, the larger, lowered shear stress lesions contained more macrophages than did lesions in the oscillatory shear stress regions. Consequently, differences in infiltration capacity of monocytes induced by lowered and oscillatory shear stress could have resulted in the observed differences in lesion size. We also found increased expression of the atherosclerosis markers IL-6 and C-reactive protein in lesions of the lowered shear stress area, indicating enhanced proatherogenic inflammatory activity. 30,31 IL-6 is an important factor for increased plaque vulnerability because it stimulates recruitment of macrophages and leukocytes and is a crucial regulator of extracellular matrix remodeling (increasing the activity of MMPs such as MMP-9). Actually, greater MMP activity was observed in lowered shear stress compared with the oscillatory shear stress regions. Macrophages have been shown to induce collagen breakdown in fibrous caps of human atherosclerotic plaques associated with cellular expression and zymographic evidence of MMP activity. 32 Increased collagen breakdown in the lowered shear stress lesions by MMP activity contributes to weakening of the fibrous cap, increasing its vulnerability to rupture. An augmented inflammatory response also causes a decline in vascular smooth muscle cells, mainly due to inhibition of proliferation by interferon- (released by leukocytes) and increased apoptosis. 18 As a result, lesions induced by lowered shear stress contain relatively fewer vascular smooth muscle cells than do lesions induced by oscillatory shear stress. Thus, lowered shear stress appears to induce larger plaques with a stronger inflammatory response, giving rise to lesions with characteristics of a vulnerable plaque. In summary, the present study has identified shear stress patterns as essential factors in atherosclerotic lesion development, size, composition, and vulnerability. Future studies are needed to elucidate the pathways by which endothelial cells initially react to shear stress during atherogenesis and will be important for understanding the development of the vulnerable plaque. With the proatherogenic shear stress fields induced by the cast, we have

9 2752 Circulation June 13, 2006 generated an animal model in which both stable and unstable lesions can be studied in 1 straight vessel segment. Intraplaque hemorrhages have been previously observed in other atherosclerosis-prone mouse models (reviewed in Rekhter 33 ). In our model, however, intraplaque hemorrhages occurred more frequently in a controlled fashion, even without additional stimuli such as angiotensin II. This will create opportunities to further study the molecular pathways involved and to evaluate therapies aimed at plaque stabilization. Acknowledgments The authors thank Monique de Waard for excellent technical assistance. Menno Buisman and Rene Thiesen are greatly acknowledged for providing us with pictures of the computational fluid dynamics. Sources of Funding This work was supported by the Netherlands Heart Foundation grant 2002T45 and the Interuniversity Cardiology Institute of the Netherlands project 33. None. Disclosures References 1. Pedersen EM, Oyre S, Agerbaek M, Kristensen IB, Ringgaard S, Boesiger P, Paaske WP. Distribution of early atherosclerotic lesions in the human abdominal aorta correlates with wall shear stresses measured in vivo. Eur J Vasc Endovasc Surg. 1999;18: Wentzel JJ, Kloet J, Andhyiswara I, Oomen JA, Schuurbiers JC, de Smet BJ, Post MJ, de Kleijn D, Pasterkamp G, Borst C, Slager CJ, Krams R. Shear-stress and wall-stress regulation of vascular remodeling after balloon angioplasty: effect of matrix metalloproteinase inhibition. Circulation. 2001;104: Davies PF, Polacek DC, Shi C, Helmke BP. The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis. Biorheology. 2002;39: Buchanan JR Jr, Kleinstreuer C, Truskey GA, Lei M. Relation between non-uniform hemodynamics and sites of altered permeability and lesion growth at the rabbit aorto-celiac junction. Atherosclerosis. 1999;143: Stone PH, Coskun AU, Kinlay S, Clark ME, Sonka M, Wahle A, Ilegbusi OJ, Yeghiazarians Y, Popma JJ, Orav J, Kuntz RE, Feldman CL. Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in-stent restenosis in humans: in vivo 6-month follow-up study. Circulation. 2003;108: Shyy JY, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res. 2002;91: Cheng C, de Crom R, van Haperen R, Helderman F, Gourabi BM, van Damme LC, Kirschbaum SW, Slager CJ, van der Steen AF, Krams R. The role of shear stress in atherosclerosis: action through gene expression and inflammation? Cell Biochem Biophys. 2004;41: Cheng C, van Haperen R, de Waard M, van Damme LC, Tempel D, Hanemaaijer L, van Cappellen GW, Bos J, Slager CJ, Duncker DJ, van der Steen AF, de Crom R, Krams R. Shear stress affects the intracellular distribution of enos: direct demonstration by a novel in vivo technique. Blood. 2005;106: Curfs DM, Lutgens E, Gijbels MJ, Kockx MM, Daemen MJ, van Schooten FJ. Chronic exposure to the carcinogenic compound benzo- [a]pyrene induces larger and phenotypically different atherosclerotic plaques in ApoE-knockout mice. Am J Pathol. 2004;164: Weiss D, Kools JJ, Taylor WR. Angiotensin II induced hypertension accelerates the development of atherosclerosis in apoe-deficient mice. Circulation. 2001;103: von der Thusen JH, van Vlijmen BJ, Hoeben RC, Kockx MM, Havekes LM, van Berkel TJ, Biessen EA. Induction of atherosclerotic plaque rupture in apolipoprotein E / mice after adenovirusmediated transfer of p53. Circulation. 2002;105: Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998;18: Gimbrone MA Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann N Y Acad Sci. 2000;902: ; discussion Cunningham KS, Gotlieb AI. The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest. 2005;85: van Haperen R, de Waard M, van Deel E, Mees B, Kutryk M, van Aken T, Hamming J, Grosveld F, Duncker DJ, de Crom R. Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. J Biol Chem. 2002;277: Lardenoye JH, Delsing DJ, de Vries MR, Deckers MM, Princen HM, Havekes LM, van Hinsbergh VW, van Bockel JH, Quax PH. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000;87: Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000;20: Lutgens E, van Suylen RJ, Faber BC, Gijbels MJ, Eurlings PM, Bijnens AP, Cleutjens KB, Heeneman S, Daemen MJ. Atherosclerotic plaque rupture: local or systemic process? Arterioscler Thromb Vasc Biol. 2003;23: Topper JN, Gimbrone MA Jr. Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype. Mol Med Today. 1999;5: Truskey GA, Barber KM, Rinker KD. Factors influencing the nonuniform localization of monocytes in the arterial wall. Biorheology. 2002;39: Dai G, Kaazempur-Mofrad MR, Natarajan S, Zhang Y, Vaughn S, Blackman BR, Kamm RD, Garcia-Cardena G, Gimbrone MA Jr. Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature. Proc Natl Acad Sci U S A. 2004;101: Ivan E, Khatri JJ, Johnson C, Magid R, Godin D, Nandi S, Lessner S, Galis ZS. Expansive arterial remodeling is associated with increased neointimal macrophage foam cell content: the murine model of macrophage-rich carotid artery lesions. Circulation. 2002;105: Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz SM. Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse. Arterioscler Thromb Vasc Biol. 2000;20: Takaya N, Yuan C, Chu B, Saam T, Polissar NL, Jarvik GP, Isaac C, McDonough J, Natiello C, Small R, Ferguson MS, Hatsukami TS. Presence of intraplaque hemorrhage stimulates progression of carotid atherosclerotic plaques: a high-resolution magnetic resonance imaging study. Circulation. 2005;111: Mazzolai L, Duchosal MA, Korber M, Bouzourene K, Aubert JF, Hao H, Vallet V, Brunner HR, Nussberger J, Gabbiani G, Hayoz D. Endogenous angiotensin II induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE / mice. Hypertension. 2004;44: Hilfiker A, Hilfiker-Kleiner D, Fuchs M, Kaminski K, Lichtenberg A, Rothkotter HJ, Schieffer B, Drexler H. Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II. Circulation. 2002;106: Morigi M, Zoja C, Figliuzzi M, Foppolo M, Micheletti G, Bontempelli M, Saronni M, Remuzzi G, Remuzzi A. Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells. Blood. 1995;85: Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res. 1998;82: Mohan S, Mohan N, Valente AJ, Sprague EA. Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. Am J Physiol. 1999;276:C1100 C1107.

10 Cheng et al Shear Stress and Atherosclerosis Paul A, Ko KW, Li L, Yechoor V, McCrory MA, Szalai AJ, Chan L. C-reactive protein accelerates the progression of atherosclerosis in apolipoprotein E deficient mice. Circulation. 2004;109: Schieffer B, Selle T, Hilfiker A, Hilfiker-Kleiner D, Grote K, Tietge UJ, Trautwein C, Luchtefeld M, Schmittkamp C, Heeneman S, Daemen MJ, Drexler H. Impact of interleukin-6 on plaque development and morphology in experimental atherosclerosis. Circulation. 2004;110: Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995;92: Rekhter MD. How to evaluate plaque vulnerability in animal models of atherosclerosis? Cardiovasc Res. 2002;54: Delsing DJ, Offerman EH, van Duyvenvoorde W, van Der Boom H, de Wit EC, Gijbels MJ, van Der Laarse A, Jukema JW, Havekes LM, Princen HM. Acyl-CoA:cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowereding effect in ApoE*3-Leiden mice. Circulation. 2001;103: CLINICAL PERSPECTIVE Atherosclerotic lesions are observed predominantly in curved arteries (like the coronary artery) and near side branches (like the carotid bifurcation), where low shear stress or vortices with oscillatory shear stress occur, suggesting a causal relation. We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stress in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or with vortices, whereas the increased shear stress regions were unaffected. Lower shear stress lesions were larger; contained fewer smooth muscle cells, less collagen, and more lipids; and showed more outward vascular remodeling than did lesions in oscillatory shear stress regions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity were higher in lowered shear stress regions. Spontaneous and angiotensin II induced intraplaque hemorrhages occurred in the lowered shear stress regions only. Our findings identify lowered shear stress as an atherogenic condition that induces the growth of plaques with a vulnerable phenotype. This type of atherosclerotic lesion has great clinical importance, because vulnerable plaques are prone to rupture, causing 70% of sudden cardiac deaths in humans. The outcome of our experiments in mice argues for further studies in vascular areas with decreased shear stress in human patients, eg, upstream from plaques causing severe stenoses. The development of vulnerable lesions may be predicted by assessing the changes in shear stress level. Also, our model creates opportunities to further study the molecular pathways involved and to evaluate therapies aimed at plaque stabilization.