Regression Of Vulnerable Plaque Progression In Rabbits By Short-term High-dose Statin

Objectives1. To establish an animal model of vulnerable atherosclerotic plaque in New Zealand white rabbits abdominal aorta.2. To evaluate the value of intravascular ultrasound in recognition of vulnerable plaque by comparison with pathological results.3. To explore the relationship between plaque stability and the lipids and the major pro-inflammatory cytokines.Materials and methodsNew Zealand rabbits aged 4 months(n=16)were randomly divided into 2 groups: fat-rich diet group(group A, n=6) and fat-rich combined with arterial intimal injury group(group B, n=10). Rabbits of group B after 2 weeks underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a fat-rich diet (1%cholesterol) for 10 weeks. Blood was drawn from rabbits fasting overnight to measure lipid profile at the beginning of the study, after week 2, the end of week12 on termination of experiment. At the same time,ELISA was used to quantify the amount of different inflammation mediators such as hs-CRP,MMP-3, IL-1, IL-10, TNF-α, and ox-LDL. Rabbits of group B underwent intravascular ultrasound studies after anesthesia on termination of experiment. Rabbits of 2 groups were sacrificed, the abdominal aorta of 5cm was cut down distal to the takeoff of the left renal artery, and 3mm specimen was embedded in paraffin, and then cut into 5um-slices and fixed on glass slides for the purpose of pathological and immunohistochemical analysis.Results1. There were 2 rabbits of group B died in accident during experiment.2. Biochemical studiesThe serum lipids levels of all rabbits increased significantly after the fat-rich diet, with further increase of levels of LDL-c and Fig on termination of the experiment (P<0.01), but values did not significantly differ between two groups (P>0.05).Levels of hs-CRP,MMP-3, IL-1, IL-10, TNF-α, and ox-LDL were no significant differences between the 2 groups at baseline and week 2(all P>0.05). However,the levels of six inflammatory biomarkers were significantly higher in rabbits of group B than those in rabbits of group A on termination of the experiment (all P<0.01). The levels of hs-CRP,IL-1, IL-10, TNF-α, and ox-LDL of all rabbits were significantly higher at week 2 than at baseline, and the levels of hs-CRP,IL-1, IL-10, TNF-α, and ox-LDL of group B increased further on termination of the experiment, as compared to group A. The level of MMP-3 was significantly higher on termination of the experiment in group B.3. VH-IVUS imaging and histologyEight rabbits of group B underwent intravascular ultrasound, a total of 276 atherosclerotic plaques were analyzed. VH-IVUS had a sensitively of 83.3% and 92.9% for the detection of non-calcified and calcified TCFA, respectively. VH-IVUS correctly identified 83.3% of the non-calcified FA and 81.3% of the calcified FA. For the detection of PIT, VH-IVUS showed a sensitivity of 81.7% and a positive predictive value of 75.4%.Linear regression analysis showed a strong correlation between histology and VH-IVUS for the percent area of fibrous tissue (R=0.845, P<0.001), fibro-fatty tissue (R=0.791, P<0.001), necrotic calcified tissue (R=0.793, P<0.001) and confluent necrotic core (R=0.731, P<0.001).The inter-observer variability of the intimal boundaries and the medial- adventitial boundaries were low. Linear regression analysis shows a strong correlation between two measurements, which were performed by two observers (R=0.993 for intimal boundaries and 0.996 for medial-adventitial boundaries).4. Histological and immunohistochemical stainingIn addition to intra-media thickness, the plaque thickness, fibrous cap thickness and plaque thickness/ intra-medial thickness ratio were different significantly between the two groups (P<0.01). The results of immunostainingα-actin were less expression in group B than group A, the positive staining of CD68 were higher in group B than group A.ConclusionsWe have developed an animal model of vulnerable plaque in New Zealand white rabbit abdominal aorta. vulnerable plaque is successfully produced by fat-rich diet together with balloon-induced endothelial injury in the rabbit abdominal aorta. Linear regression analysis showed a strong correlation between histology and VH-IVUS, VH-IVUS is a feasible, reproducible and valuable means of the vulnerable plaque identification. Objectives1. To test the hypothesis that short-term high-dose pitavastatin intervention has anti-atherosclerotic progression and plaque-stabilizing effect in a rabbit model of vulnerable plaques.2. To elucidate the mechanisms of short-term high-dose pitavastatin treatment in stabilizing vulnerable plaques. Methods1. Anmal experiment protocolA rabbit model of vulnerable plaque was produced by the method in part I. The male New Zealand white rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed with a high-cholesterol diet (l% cholesterol) for 10 weeks. A total of 24 male New Zealand white rabbits were identified vulnerable plaques in abdominal aorta by VH-IVUS. Then rabbits were randomly divided into 3 groups: group A was served as controls (without treatment, n=8), group B was given usual dose pitavastatin (1mg/kg/d, n=8), group C was given high-dose pitavastatin (10mg/kg/d, n=8). One week later, all rabbits underwent VH-IVUS examination.2. Biochemical studiesBlood was drawn from rabbits fasting overnightat the end of week12 and 13 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MMP-3, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL.3. Intravascular ultrasound studiesVH-IVUS was accomplished at the end of week 13 to measure the external elastic membrane area(EEMA), the lumen area(LA),plaque area(PA),the percentage of plaque burden(PB). VH-IVUS identified four different plaque components (fibrous, fibrofatty, necrotic and calcified tissue).4. Histological and immunohistochemical stainingSerial cross-sections underwent general histological staining with hematoxylin & eosin(HE), picrosirius red, Masson and specific immunohistochemical stainings. The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,andα-smooth muscle cell actin to detect VSMCs. The fibrous cap thickness, the intra-media thickness, the plaque thickness, and the plaque thickness/ intra-medial thickness ratio were measured and values averaged. VSMCs and macrophages were expressed as a percentage of statining area divided by plaque area.5. Quantitative Real-time reverse transcriptase-polymerase chain reactionThe mRNA expression of various inflammation medicators LOX-l, MMP-3, MMP-9, TIMP-1 and TIMP-2 in the abdominal arterial atherosclerosis lesions was determined by RT-PCR. Results1. Biochemical changesThe levels of TC, TG and LDL-C were decreased significantly in pitavastatin groups (P<0.01), and HDL-C was increased in high-dose pitavastatin group(P<0.01). At the week 13, the levels of TC, TG and LDL-C in pitavastatin group were lower than those in control group, but only HDL-C in high-dose pitavastatin group was higher than that in usual dose pitavastatin group (P<0.01).The levels of MMP-3, hs-CRP, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL were decreased significantly after treatment in pitavastatin groups (P<0.05 for TNF-α,hs-CRP, MMP-9 in usual dose group and P<0.01 for IL-1, IL-10, MMP-3, ox-LDL in usual dose group and all in high dose group). The levels of MMP-3, hs-CRP, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL in pitavastatin groups was lower than those in control group, and the levels of MMP-3, hs-CRP, TNF-α, IL-1 and IL-10 in high-dose pitavastatin group was lower than those in usual dose group(P<0.01 for MMP-3, hs-CRP,TNF-α, IL-1, IL-10 ). 2. Intravascular ultrasound alterationsThe lumen area was increased significantly (P=0.01), and the plaque area and the plaque burden were decreased significantly after pitavastatin treatment in high-dose group (P<0.01), however, fibro-fatty volume and fibro-fatty (%) was decreased significantly after the treatment in high-dose group. Fibro-fatty volume in high-dose group was decreased significantly in control and usual dose group (P<0.01 and P<0.05).3. Histological changesPlaque thickness in high-dose pitavastatin group was decreased significantly than in control and usual dose groups (P<0.05), however, fibrous cap was increased significantly (P<0.01). Lower macrophage and higher SMCs were found in high-dose pitavastatin group than in control and usual dose groups (P<0.01).4. Quantitative Real-time RT-PCRThe relative mRNA expressions of LOX-l,MMP-3,MMP-9 were lower in treatment groups than in control group (all P<0.01), and high dose group was decreased significantly than usual dose group (P<0.01). On the contrary,TIMP-1 and TIMP-2 mRNA expressions were higher in treatment groups than in control group, and high dose group was increased significantly than usual dose group.Conclusions1. Short-term pitavastatin treatment is effective in lipid lowering with a dose-dependent way.2. Short-term pitavastatin treatment has anti-inflammation effect with a dose-dependent way.3. Short-term with high-dose pitavastatin intervention enhances the vulnerable plaque stability and inhibits the plaque progression.4. The mechanisms involved in stabalising vulnerable plaques by short-term high-dose pitavastatin may be mainly due to its lipid lowering, anti-inflammation and anti-oxidative effects.