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Integrative Physiology/Experimental Medicine

Endogenous Renovascular Hypertension Combined With Low Shear Stress Induces Plaque Rupture in Apolipoprotein E–Deficient Mice

Shu-xuan Jin, Ling-hong Shen, Peng Nie, Wei Yuan, Liu-hua Hu, Dan-dan Li, Xue-jin Chen, Xiao-kun Zhang, Ben He
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https://doi.org/10.1161/ATVBAHA.111.236158
Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2372-2379
Originally published September 12, 2012
Shu-xuan Jin
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Ling-hong Shen
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Peng Nie
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Wei Yuan
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Liu-hua Hu
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Dan-dan Li
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Xue-jin Chen
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Xiao-kun Zhang
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Ben He
From the Department of Cardiology, Renji Hospital (S.J., L.S., P.N., W.Y., L.H., D.L., B.H.), and Department of Laboratory Animal Science (X.C.), Shanghai Jiaotong University School of Medicine, Shanghai, China; and Cancer Center (X.Z.), Sandford-Burnham Medical Research Institute, La Jolla, CA.
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Abstract

Objective—The development of a murine model of spontaneous atherosclerotic plaque rupture with luminal thrombus.

Methods and Results—Combined partial ligation of the left renal artery and left common carotid artery in 8-week-old apolipoprotein E–deficient mice induced endogenous renovascular hypertension and local low oscillatory shear stress in the left common carotid artery. After 8 weeks, a fresh left common carotid artery lumen thrombus associated with severe plaque burden was found in 50% (10/20) of the mice. Histological analyses indicated that all left common carotid artery lesions had vulnerable features, and 50% (5/10) of the mice showed plaque rupture with a lumen thrombus. Multiple layers with layering discontinuity and intraplaque hemorrhages were found in 80% (8/10) of the mice. Further experiments showed that both increased blood pressure, and angiotensin-II contributed to plaque progression and vulnerability. Decreased intimal collagen associated with increased collagenase activity and matrix metalloproteinase expression also resulted in plaque disruption.

Conclusion—We demonstrate a murine model of spontaneous plaque rupture with a high incidence of luminal thrombus. The model not only nicely recapitulates the pathophysiological processes of human plaque rupture but it is also simple, fast, and highly efficient to generate.

  • animal model
  • plaque rupture
  • thrombus
  • mechanical stress
  • angiotensin

Introduction

The concept of plaque rupture was first reported in 1844,1,2 and researchers had not demonstrated the connection between plaque stability with coronary thrombus until the mid-20th century.3,4 Today, it is widely accepted that vulnerable plaque, defined as a lesion consisting of a necrotic core with an overlying thin ruptured fibrous cap, is the leading cause of coronary luminal thrombosis.5,6 The disruption of the cap allows contact of the blood in the lumen of the artery with the highly thrombogenic material of the necrotic core. The activated clotting cascade that follows results in coronary thrombus rich in aggregated platelets and fibrin. Plaque rupture reportedly contributes to 75% of the thrombi responsible for acute coronary syndromes and is, therefore, of great importance in understanding how the atherosclerotic lesions convert from stable to vulnerable phenotypes.7

A suitable animal model bearing close resemblance to the pathophysiological processes of plaque rupture would be useful for investigating potential therapeutic interventions. Based on the definition of fibroatheroma with cap disruption and luminal thrombus that communicates with the underlying necrotic core for plaque rupture,7 the presence of luminal thrombus should be regarded as a key diagnostic feature of plaque rupture in mice. Given the superiority of mice compared with other animals, tremendous efforts have been devoted to generating a murine model of plaque rupture particularly associated with thrombosis. In recent years, breakthrough occurred after apolipoprotein E (ApoE) knockout mice emerged.8,9 Varied manipulations tested to induce lesion progression and plaque instability have seldom shown convincing evidence of platelet- and fibrin-rich occlusive thrombus formation at the presumed rupture site without mechanical, physical, or chemical triggers.10–12 The reasons for failure were presumed to be small vessel size, enhanced fibrinolytic function, and mildly occlusive lesions in mice.13–15 As a result, Jackson et al16 suggested that acute plaque rupture in mice might be defined as a visible defect in the fibrous cap accompanied by intrusion of erythrocytes into the plaque below it. Buried fibrous caps in murine lesions might also be considered evidence of old plaque rupture. However, plaque hemorrhage and buried fibrous caps being just indirect indicators of plaque rupture, there is an urgent need for a suitable model in which spontaneous plaque rupture occurs with visible luminal thrombus.

Therefore, in this study, using the method of partial ligation of left carotid and left renal arteries, we induced local stress change, as well as continuously activated renin–angiotensin system in ApoE knockout mice. High incidence of spontaneous plaque rupture associated with lumen thrombosis was successfully generated by combining systematic and local factors. Furthermore, this simple murine model not only nicely recapitulates the pathophysiological processes of human plaque rupture but also has rapid plaque progression.

Materials and Methods

Mice

ApoE-deficient (ApoE−/−) mice on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME). Age- (8-week-old) and sex-matched animals weighing between 20 and 25 g were enrolled in the study. Mice batch 1 was randomly assigned to 5 groups: combined partial ligation of left renal artery and left common carotid artery (LCCA) (R+C), partial ligation of left renal artery (R), partial ligation of LCCA (C), sham control (S), and open control (O). Mice batch 2 was also randomly assigned to 5 groups: combined partial ligation of left renal artery and LCCA + intragastrically administered normal saline (R+C+NS), combined partial ligation of left renal artery and LCCA + intragastrically administered losartan (R+C+LO), partial ligation of LCCA + subcutaneous normal saline infusion (C+NS), partial ligation of LCCA + subcutaneous angiotensin II infusion (C+ANG), and partial ligation of LCCA + subcutaneous phenylephrine infusion (C+PHE). For time series experiment, mice were randomly assigned to 4 groups: sham operation was performed in mice of group 1; mice of other groups underwent partial ligation both of LCCA and left renal artery and euthanized at 2 weeks, 4 weeks, and 8 weeks after surgery. All surgeries were performed under the dissecting microscope. Mice were provided with a standard rodent diet and tap water ad libitum during the experiment. All animals were euthanized 8 weeks after the procedure. All animal work was performed in accordance with the guidelines on animal care of Shanghai Jiaotong University School of Medicine.

Animal Surgery

Partial Carotid Ligation

Partial ligation of the LCCA was performed, as described previously.17 Briefly, mice were anesthetized using intraperitoneal injection of pentobarbital sodium (50 mg/kg) and maintained at 37°C on a heating pad. After blunt dissection to expose the distal branches of LCCA, blood flow was reduced by ligation (6-0 silk) of all branches of LCCA except for the left thyroid artery (Figure 1A). After validation that blood flow was present, the incision was closed with a suture. Sham ligation consisted of suture placement without ligation.

Figure 1.
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Figure 1.

Combined partial ligation of the left renal artery and left common carotid artery (LCCA) causes renovascular hypertension and low flow. A, Schematic diagram of the combined partial ligation of the left renal artery (LRA; left) and left coronary artery (LCA; right) in 8-week-old apolipoprotein E knockout mice. Partial ligation of the left common carotid artery (LCCA) was generated by ligation of its 3 branches; the left internal carotid artery (LICA), left occipital artery (LOA), and left external carotid artery (LECA), leaving the left superior thyroid artery (LSTA) patent. Pictures of the pin gauge, ligation under dissecting microscope, and representative images of tissue collection are shown from upper left to right. B, Representative kidney images at 8 weeks after the procedure (scale bar=5 mm). Kidneys of euthanized animals were harvested and photographed after paraformaldehyde perfusion. The longitudinal diameter ratio between the left and right kidneys was then measured (*P<0.05 vs group O; n=3). Systolic blood pressure was measured before ligation and at 4 weeks (*P<0.05 vs preligation; n=12–20) and 8 weeks (#P<0.05 vs preligation; n=12–20) after ligation. C, Ultrasound showing flow velocity profiles before and 4 weeks after ligation. Doppler images of blood flow in the LCCA is representative of at least 10 mice deficient in apolipoprotein E. Mean blood flow in the LCCA significantly decreased in groups C and R+C (*P<0.05 vs preligation; n=3). Peak systolic velocity significantly increased in groups R and R+C (*P<0.05 vs preligation; n=3), and decreased in group C (*P<0.05 vs preligation; n=3). Values are mean±SEM. R indicates partial ligation of left renal artery; C, partial ligation of LCCA; S, sham control; O, open control. Original magnifications are ×200 (A, B) and ×400 (C).

Partial Ligation of Left Renal Artery

Endogenous renovascular hypertension was generated by modification of the 2K1C murine model previously described.18,19 Briefly, a spacer instead of a clip was chosen to ligate the left renal artery to generate a precise steady stenosis. Mice were anesthetized and the left kidney was exposed through a small flank incision. After isolation by blunt dissection, the left renal artery was tied off (6-0 silk) along with a spacer (pin gauge, outer diameter=0.11 mm, Figure 1A). The pin gauge was subsequently pulled out leaving a tight stenosis in the artery. The kidney was then gently pushed back into the retroperitoneal cavity. The muscle layer and the skin incision were closed with sutures. The pin gauge is commercially available (Jining Hengsheng Precise Measuring and Cutting Co Ltd, Shandong, China), and its size was verified using a micrometer caliper. A sham procedure including the entire surgery without ligation was applied in control mice. Successful induction of renovascular hypertension was defined as systolic blood pressure >120 mm Hg 2 weeks after the procedure. Mice that failed to meet this criterion were excluded from the study.

Detailed descriptions of the materials and experimental methods are available in the online-only Data Supplement.

Results

No animals died during this experiment. There were no differences in body weight, cholesterol levels, and renal function detected among the groups (see Tables II and III in the online-only Data Supplement).

Combined Partial Ligation of the Left Renal Artery and LCCA Causes Renovascular Hypertension and Low Shear Stress

According to the original work by Wiesel et al,18 2K1C ApoE−/− mice were considered a suitable model for endogenous renovascular hypertension because of a continuously activated renin–angiotensin system. In this model, renal perfusion was reduced using a clip with lumen size 0.12 mm, which induced high blood pressure without producing renal infarction. Because the clip was unavailable to us, we modified this model by partial unilateral renal artery ligation using a pin gauge. In preliminary experiments, pin gauges with outer diameters ranging from 0.09 to 0.13 mm were used to induce renovascular hypertension (data not shown), and the outer diameter of 0.11 mm met the definition of hypertension (see Materials and Methods). As shown in Figure 1B, significant shrinkage of the ligated left kidney was observed in groups R and R+C with left/right kidney ratio decreased almost by half compared with the other groups. Systolic blood pressure of groups R and R+C was elevated 1 week after the procedure, reached maximal values at 4 weeks (139±2 mm Hg for group R and 143±1.7 mm Hg for group R+C), and then decreased, but remained higher than the initial level until euthanization.

Next, flow velocity in the LCCA was determined by ultrasonography 4 weeks after the procedure. Compared with preligation, peak systolic velocity (Vmax) significantly increased in groups R (121.9±1.7 cm/s versus 222±11.2 cm/s; P<0.05) and R+C (124±3.5 cm/s versus 205.4±15.6 cm/s; P<0.05), but decreased in group C (115.1±5.3 cm/s versus 43.2±2.4 cm/s; P<0.05; Figure 1C). Mean blood flow (MBF) was slightly elevated in group R (0.66±0.01 mL/min versus 0.96±0.12 mL/min; P=0.07), whereas it was strikingly reduced in group C (0.71±0.07 mL/min versus 0.06±0.003 mL/min; P<0.05) and group R+C (0.67±0.02 mL/min versus 0.28±0.05 mL/min; P<0.05). Vmax was significantly increased in group R and decreased in group C compared with groups O and S (P<0.05), but there was no difference between the group R+C and the controls (P=0.08). The MBF in groups C and R+C was significantly decreased compared with controls (P<0.05), with no difference between group C and R+C (P=0.13). An increased MBF was seen in group R compared with the other 4 groups, but this did not reach statistical significance.

It is thus evident that partial ligation of the left renal artery increases MBF and Vmax in the LCCA, which resulted in higher shear stress. However, partial ligation of the LCCA reduces MBF and Vmax and contributes to a low oscillatory shear stress. With combined partial ligation, blood flow is similarly disturbed, but elevated peak velocities might lead to higher peak circumferential stress, which may contribute to increased occurrence of plaque disruption.

Combined Renovascular Hypertension and Low Shear Stress Results in Accelerated Atherosclerosis and Plaque Disruption Associated With Lumen Thrombosis in ApoE−/− Mice

To determine whether the combined partial ligation of the left renal artery and LCCA causes spontaneous plaque rupture and lumen thrombosis in ApoE−/− mice, the LCCA was isolated after pressure perfusion. Fresh LCCA lumen thrombus associated with severe plaque burden was found in 50% (10/20) of the mice in group R+C and 10.5% (2/19) in group C (P<0.05; group C versus group R+C). The LCCA in the other groups lacked thrombus (Figure 2A). The plaque phenotype was then examined by histological analysis after serial sections. As shown in Figure 2A, groups O and S did not develop atherosclerotic lesions. Group R showed stable, intermediate lesions with increased intimal surface area compared with group S (5103±635 μm2 versus 2228±193 μm2, respectively; P<0.05). Advanced atherosclerotic lesions were observed in groups C and R+C, with intimal surface area increased nearly 20× and 53× compared with group S, respectively (45 841±6995 μm2, P<0.05 and 117 812±16 517 μm2, P<0.05, respectively, compared with 2228±193 μm2; Figure 2D). Smooth muscle cell α-actin immunoreactivity indicated increased actin-positive areas in the intima of group C compared with controls (36.9±2.0% versus 18.9±3.9%; P<0.05; Figure 2E), but actin-positive areas were significantly less in group R+C compared with group C (13.3±1.1% versus 36.9±2.0%; P<0.05). The number of proliferating cell nuclear Ag–positive smooth muscle cells per lesion area in group C was more than that in group R+C (26.8±4.4 versus 14.6±1.5; P<0.05; Figure IB in the online-only Data Supplement). The apoptosis index of smooth muscles cells in group R+C showed an increasing trend compared with group C, but it did not reach statistical significance (see Figure IC in the online-only Data Supplement). Significantly increased macrophage-positive areas were also seen in the intima of mice in groups R, C, and R+C compared with group O. The macrophage-positive areas in group R+C were higher than those in group C (38.5±2.4% versus 28.0±2.0%, respectively; P<0.05; Figure 2F). However, there was no difference in the macrophage proliferation and apoptosis index between group C and R+C (see Figure ID and IE in the online-only Data Supplement).

Figure 2.
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Figure 2.

Combined partial ligation of the left renal artery and left common carotid artery (LCCA) rapidly induced advanced atherosclerotic lesions with plaque disruption associated with lumen thrombosis. A, LCCA of apolipoprotein E knockout mice 8 weeks after the procedure. Shown from left to right columns are representative gross images of 12 to 20 mice (scale bar=2 mm), hematoxylin-eosin staining, and immunostaining for α-smooth muscle cell (SMC) actin and macrophages (scale bar=100 μm). B, Intraplaque hemorrhage was observed in lesions with mature, intact fibrous cap and necrotic core containing red blood cells and fibrin. C, Plaque rupture (thin arrow) with nearly occlusive lumen thrombus (T), and neovessels (asterisk) were observed in group R+C. D, Intimal surface area of atherosclerotic lesions was quantified for each group (*P<0.05 vs sham control [S] group; n=6–10). E, α-SMC actin quantification (*P<0.05 vs open control [O] group; n=10). F, Macrophage quantification (* P<0.05 vs group O; n=6–10). Values are mean±SEM. R inidicates partial ligation of left renal artery; C, partial ligation of LCCA.

Further characterization of the lesion phenotype showed that 70% (7/10) of lesions in group C and all (10/10) lesions in group R+C had vulnerable features. Multiple layers with associated discontinuity of layering, indicative of plaque instability, were also found in groups C and R+C. Interestingly, there was a significant increase of intraplaque hemorrhage in group R+C compared with group C (80% versus 10%, respectively; P<0.05), accompanied by an increasing trend of plaque rupture with lumen thrombus (50% versus 10%; P=0.07). Examples of intraplaque hemorrhage and plaque rupture with luminal thrombus are shown in Figure 2B and 2C, and the data are summarized in the Table.

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Table.

Lesion Features

Moreover, a time series experiment was performed to determine atherosclerosis progression at different time points. Increased left carotid artery intimal surface area was observed 2 weeks after combined ligation. At 8 weeks, average intimal surface area elevated to more than 3× that of 2 weeks, and lumen cross-sectional area decreased by >75% in all animals except in mice with occlusive thrombus (see Figure II in the online-only Data Supplement). Vulnerable phenotypes were observed from 4 weeks after surgery, and all animals displayed lesions with unstable features at 8 weeks. At the same time, high incidences of intraplaque hemorrhage and plaque rupture, 83.3% and 66.7%, respectively, were also observed at 8 weeks. These data show that plaque progression seems to reach a peak around the 8-week time point (see Table IV in the online-only Data Supplement).

In summary, activating the endogenous renin–angiotensin system led only to the development of stable, intermediate lesions in ApoE−/− mice. Modification of local shear stress in the carotid artery led to accelerated atherosclerosis and complex lesions with only a minor incidence of thrombosis. However, the combination of these 2 stimulating factors in ApoE−/− mice resulted in advanced atherosclerotic lesions with plaque rupture and a high incidence of lumen thrombus.

Increased Blood Pressure and Angiotensin-II Both Contribute to Atherosclerotic Progress and Plaque Vulnerability Under Low Stress Shear

To substantiate the role of endogenous angiotensin-II as an inducer of plaque progression in carotid artery exposed to low wall shear stress, mice were treated with losartan, which blocks type 1 angiotensin II (AT1) receptors, before combined partial ligation of the left renal artery and LCCA. In these mice, there was no significant increase of heart rate and blood pressure at either 4 weeks or 8 weeks postsurgery (Figure 3B and 3C ). Moreover, atherosclerosis extension was prevented, and intimal surface area was close to that observed in animals that underwent LCCA partial ligation alone (Figure 3A and 3D). Plaque morphology was also comparable with that of the carotid artery with low stress shear alone, in that the same rate of plaque rupture with luminal thrombus was observed (see Table V in the online-only Data Supplement).

Figure 3.
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Figure 3.

Role of blood pressure and angiotensin-II in plaque progression and vulnerability under low stress shear. Representative images of plaque progression in group R+C+NS, group R+C+LO, group C+NS, group C +ANG, and group C+PHE with hematoxylin-eosin staining (x200 magnification; scale bar=100 μm; A). Heart rate (HR) (B) and systolic blood pressure (SBP) (C) were observed before and 4 weeks and 8 weeks after surgery (*P<0.05 vs group C+NS, n=6-10). Intimal surface area of atherosclerotic lesions (D) and macrophage quantification (E) were quantified for each group (*P<0.05 vs group R+C+NS; #P<0.05 vs group C+NS; n=6–10). R indicates partial ligation of left renal artery; C, partial ligation left common carotid artery; NS, intragastrically administered normal saline; LO, intragastrically administered losartan; ANG, subcutaneous angiotensin II infusion; PHE, subcutaneous phenylephrine infusion.

Next, we studied the contribution of angiotensin II and blood pressure in plaque progression and vulnerability in arteries exposed to low wall shear stress. After LCCA partial ligation, exogenous low-dose angiotensin-II was used to generate ApoE−/− mice with a high concentration of angiotensin-II and normal blood pressure, whereas phenylephrine was used to induce hypertensive mice with normal levels of angiotensin-II (Figure 3C). As shown in Figure 3A and 3D, advanced lesions were observed in groups C+ANG and C+PHE where intimal surface area increased slightly compared with group C+N (66 997±7887 μm2 P<0.05 and 59 804±2935 μm2, P<0.05, respectively, compared with 45 007±3484 μm2). But plaque area in groups C+ANG and C+PHE was still significantly less than group R+C+N (113 738±6604 μm2; P<0.05). CD68-positive areas in the intima also showed a similar pattern, and no difference was observed between group C+ANG and R+C+N (37.6±3.8% versus 42.0±2.2%; P=0.85). Lesion phenotype detection further confirmed that both increased blood pressure and angiotensin-II contribute to plaque vulnerability (see Table V in the online-only Data Supplement).

Downregulated Intimal Collagen Is Associated With Increased Collagenase Activity in Low Shear Stress Lesions of ApoE−/− Mice With Renovascular Hypertension

Because plaque rupture was observed in groups C and R+C, we evaluated the neointimal collagen content in these groups. As shown in Figure 4A, the collagen/intima ratio in group R+C was significantly lower than that in group C (5.9±1.1% versus 22.9±5.9%, respectively; P<0.05). Accordingly, an upregulated collagenase activity/intima ratio was observed in group R+C as compared with group C (25.8±5.0% versus 11.6±1.4%, respectively; P<0.05; Figure 4B).

Figure 4.
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Figure 4.

Collagen content and collagenase activity in the neointima. A, Representative images of intimal collagen content in groups C and R+C with picrosirius red staining (scale bar=100 μm). Quantification of collagen content of collagen/intima ratio in groups C and R+C (*P<0.05 vs group C; n=10; values are mean±SEM). B, Representative images and quantification of in situ collagenase activity in group C and R+C (scale bar=100 μm; *P<0.05 vs group C; n=3; values are mean±SEM). C indicates partial ligation left common carotid artery; R partial ligation of left renal artery.

Activated collagenases of the matrix metalloproteinase (MMP) family, including MMP-1, MMP-8, and MMP-13, are usually considered to initiate the first step of collagen degradation. Therefore, we examined MMP-8 and MMP-13 expression in the vascular tissue of the LCCA. As shown in Figure 5A and 5B, the mRNA levels of MMP-8 and MMP-13 were markedly increased in groups C and R+C compared with the control groups. MMP-8/intima and MMP-13/intima ratios were increased ≈2-fold in group R+C compared with group C (31.7±4.2% versus 15.4±2.4%, P<0.05 for MMP-8; and 44.1±4.1% versus 19.7±2.8%, P<0.05 for MMP-13, respectively, Figure 5C and 5D). Further analysis by coimmunofluorescence labeling of MMPs, macrophages, and vascular smooth muscle cells showed that the overexpressed MMP-8 and MMP-13 primarily costained with macrophages (Figure 5E and 5F).

Figure 5.
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Figure 5.

Expression of matrix metalloproteinase (MMP)-8 and MMP-13 in the neointima. MMP-8 (A) and MMP-13 (B) mRNA expression in vascular tissue was measured by real-time polymerase chain reaction (n=3). Representative images and quantification of MMP-8 (C) and MMP-13 (D) in group C and R+C (scale bar=100 μm; *P<0.05 vs group C; n=10). Frozen sections of mice left common carotid artery containing atherosclerotic plaques were stained with anti–MMP-8, anti–MMP-13, anti–α-actin, anti-CD68, or control IgG. Both MMP-8 (E) and MMP-13 (F) were mainly colocalized with CD68. O indicates open control; S, sham control; R, partial ligation of left renal artery; C, partial ligation of left common carotid artery.

Previous studies have shown that MMP inhibitors also contribute to extracellular matrix integrity and plaque vulnerability.20 In this model, the mRNA levels of matrix protease inhibitors as measured by real-time polymerase chain reaction also showed some increases in tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, tissue inhibitor of metalloproteinase-3, and tissue inhibitor of metalloproteinase-4 gene expression in atherosclerotic lesions of mice in group C, R, and R+C (see Figure III in the online-only Data Supplement).

Discussion

The renin–angiotensin system and local shear stress changes both contribute to plaque progression.21–23 Therefore, we hypothesized that the combination of these changes might contribute to plaque rupture. Using a partial ligation technique to induce endogenous renin–angiotensin system activation and local shear stress modification, we have generated a novel model of plaque rupture in ApoE−/− mice. The model shows spontaneous plaque rupture with a high incidence of luminal thrombus. Combined partial ligation of the left renal artery and LCCA in ApoE−/− mice after 4 weeks on a standard rodent chow induced endogenous renovascular hypertension and local low oscillatory shear stress. Eight weeks after surgery, severe plaque burden was detected in all animals, which is a vulnerable lesion that contained a number of macrophages. A high incidence of intraplaque hemorrhage and buried fibrous caps were also observed in these lesions. Furthermore, half the animals displayed fresh luminal thrombus associated with plaque rupture. Decreased collagen content was observed in the intima, which was accompanied by an increase in the expression of MMP-8 and MMP-13 and collagenase activity.

A previous study demonstrated endogenous angiotensin II–induced atherosclerotic plaque vulnerability in hypertensive hypercholesterolemic ApoE−/− mice, whereas hypertensive normal angiotensin-II mice only developed a stable phenotype.20 To substantiate the roles of angiotensin-II and blood pressure in plaque vulnerability of arteries exposed to low wall stress shear, ApoE−/− mice were administered exogenous low-dose angiotensin-II or phenylephrine after LCCA partial ligation. Both increased blood pressure, and angiotensin-II contributes to atherosclerotic progress and plaque vulnerability. The role of endogenous angiotensin-II as an inducer of plaque progression was further confirmed by intragastrically administered losartan after combined ligation, which showed similar intimal surface area to that observed in animals that underwent LCCA partial ligation alone.

One can see that present model is simple, fast, and efficient. However, the most important advantage of this model rests in the development of spontaneous plaque rupture with a high incidence of luminal thrombus, which nicely recapitulates the pathophysiological processes of human atherosclerosis. Previous studies have also observed spontaneous plaque rupture in ApoE−/− mice. However, these events occurred only in older mice after feeding with a high-fat diet for long periods (12–15 months).23–26 Sasaki et al27 have reported a simple method of atherosclerotic plaque rupture in ApoE−/− mice that used a polyethylene-cuff placement after 4 weeks of total ligation of the carotid artery. Dramatic disruption of neointima and occlusive thrombus formation were detected in the carotid artery 7 days after the cuff placement. The cuff placement eliminated alternative oxygen supply via vasa vasorum, which might lead to severe site-specific hypoxia and ischemic necrosis. Some view this plaque rupture as acute and nonspontaneous.28 Others have reported that the plaque rupture was induced by mechanical, physical, or chemical injuries in mice.11,29–31

There are several limitations to this model. First, detailed local shear stress was not analyzed in this study. Several novel techniques were developed to map localized shear stress and strain spots in human carotid or coronary arteries, including magnetic resonance imaging and intravascular ultrasound in combination with computational fluid dynamics. However, attaining accurate data using micro-ultrasound imaging systems is extremely difficult because of the size of mice carotid arteries and rapid lesion protrusion in the lumen. Thus, the exact mechanisms involved in plaque rupture in this model need to be further elucidated. For instance, the existence of plaque neovascularization, which might be important for intraplaque hemorrhage, was not examined in this model. It is also not clear what role the endothelium plays in plaque disruption in this model.

In conclusion, this study demonstrates a murine model that mimics human plaque rupture. Methods adopted in this study are simple, fast, and highly efficient. We believe that this model will be helpful in elucidating the mechanisms of human plaque rupture and assessing various therapeutic interventions in the future.

Sources of Funding

This work was supported by grant numbers 30971185, 81070239, and 81170192 from the National Natural Science Foundation; grant numbers 09JC1409400, 10JC1409400, 11JC1407100, and 10410701200 from the Shanghai Municipal Natural Science Foundation; and grant number BC103870 from the US Army Medical Research and Material.

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.111.236158/-/DC1.

  • Received August 4, 2011.
  • Accepted August 3, 2012.
  • © 2012 American Heart Association, Inc.

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    Endogenous Renovascular Hypertension Combined With Low Shear Stress Induces Plaque Rupture in Apolipoprotein E–Deficient Mice
    Shu-xuan Jin, Ling-hong Shen, Peng Nie, Wei Yuan, Liu-hua Hu, Dan-dan Li, Xue-jin Chen, Xiao-kun Zhang and Ben He
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2372-2379, originally published September 12, 2012
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    Shu-xuan Jin, Ling-hong Shen, Peng Nie, Wei Yuan, Liu-hua Hu, Dan-dan Li, Xue-jin Chen, Xiao-kun Zhang and Ben He
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2372-2379, originally published September 12, 2012
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