| BackgroudAtherosclerotic plaque rupture is the major cause of acute cardiovascular events. A plaque with a thin cap,a large lipid core,and abundant activated macrophages has been generally regarded as unstable and vulnerable to rupture.Early detection and prevention would be the only way to reduce the risk of this catastrophic life-threatening event.Studies of factors of promoting plaque unstable as well as changing plaque characteristics from unstable to stable are crucial for prevention of cardiovascular disease.Efforts to elucidate the mechanisms of vulnerable plaque as well as the studies of prevention and treatment would be greatly aided by the availability of an animal model.However,an animal model of vulnerable plaques mimicking human lesions is still lacking,which has limited the progress of basic research in plaque instability.In our previous studies,we used a catheter to inject Ad5-CMV.p53 into an abdominal aortic segment which was rich in plaques and ligated at its both ends for 10 minutes.Although we achieved a high rate of plaque rupture in rabbits,this model has several limitations which requires further improvement:First,aortic ligation may lead to abdominal organ ischemia and reperfusion injury contributing to a relatively high mortality rate;second,the transfection efficiency is difficult to control during 10 minutes' contact of Ad5-CMV.p53 suspension with the endothelium;third,p53 suspension spreads to the whole cardiovascular system after release of the aortic ligation,which may cause systemic adverse effects.Therefore,the present study was carried out to develop a new animal model of vulnerable plaques and investigate the possible mechanisms of exogenous p53-induced plaque instability using this new model.Objectives1.To estabilish an animal model of vulnerable plaques histologically identical to humans,easy to detect,and suitable to evaluate the therapeutic effect of medicine.2.To elucidate the pathological and molecular mechanisms of exogenous p53-induced plaque instability.Materials and methods1.Animal protocolAdult New Zealand White male rabbits(n=40)underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1%cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 3 groups:group A(n=16),group B(n=16)and group C(n=8).Rabbits of group C were euthanized for histological and immunohistochemical studies at the end of week 16. At week 16 since the beginning of the experiment,the other rabbits underwent intravascular ultrasound(IVUS)studies after anesthesia.After the largest plaque in the abdominal aorta was imaged with IVUS,a median incision in the abdomen was made and the corresponding portion of the abdominal aorta was isolated.Under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53(8×10~9pfu/ml)or lac Z(8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in the Ad5-CMV.p53 transfected group or Ad5-CMV.lac Z transfected group,respectively.To observe the efficiency of transfection,3 rabbits in each group were euthanized.According to Constantinides' method,two weeks after transfection,plaque disruption was triggered by administration of Chinese Russell's viper venom(0.15 mg/kg,intraperitoneally),followed 30 min later by histamine (0.02 mg/kg intravenously)at 24h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight to measure lipid profile at the beginning of the study,the end of week 10 on termination of high cholesterol diet and the end of week 16 before adventitial injection.At the same time,ELISA was used to quantify the amount of different inflammation mediators such as hs-CRP,MCP-1, sICAM-1,sVCAM-1 and ox-LDL.3.Histological and immunohistochemical stainingX-Gal staining was performed on 5μm-thick cryostat sections using aβ-galactosidase activity assay kit.Apoptosis was assessed by terminal deoxynucleotidyl transferase end-labelling(TUNEL)staining.The apoptosis rate was expressed as the proportion of apoptotic cells to total number of cells in a given area. Serial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),Masson's trichome and picrosirius red,and specific immunohistochemical staining.Slides were stained with antibodies against recombinant human p53,a macrophage-specific antibody,α-smooth muscle cell actin and tumor necrotic factor-α(TNFα).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as loss of fibrous-cap continuity with overlying luminal thrombosis.Six cross-sections from the histological sections of aorta were used for analysis of cap thickness,intima thickness and cap/intima ratio,and the values averaged.Slides were scanned by microscope for computerized image analysis with Image-Pro Plus 5.0.4.Statistical analysisValues were expressed as mean + SD and assessed by one-sample Kolmogorov-Smimov test whether it is normal distribution.Differences in continuous variables between two groups were assessed by unpaired t-test,and comparison among multiple groups was performed by analysis of variance with ANOVA.Categorical variables were analyzed by Fisher's exact test.A bivariate correlation was used to choose quantitative variables which demonstrated significant correlations with plaque rupture,and these variables were then introduced into a binary logistic regression model to select variables which had independent effects on plaque rupture.Subsequently,linear regression analyses were performed to evaluate the relationship between apoptosis and inflammatory markers including hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.A p value＜0.05 was considered statistically significant.ResultsAll animals underwent initial balloon injury and the next abdominal operation without complications and showed full recovery.1.Biochemical studiesThe serum TC,TG and LDL-C levels of all rabbits increased significantly after the high-cholesterol diet and were reduced 6 weeks later(p＜0.01).However,values did not significantly differ among the three groups(p＞0.05).Levels of hs-CRP,MCP-1,sICAM-1,sVCAM-1 and ox-LDL were not significantly different among rabbits in the three groups at baseline,week 10 and week 16 before gene transfection(all p＞0.05).However,the five inflammatory biomarkers were significantly higher in rabbits with Ad5-CMV.p53-treated group than those in rabbits with Ad5-CMV.lac Z-treated group at week 18(all p＜0.01).2.IVUS imagingBy IVUS imaging,all plaques were characterized as fibrofatty.No ruptured plaque and thrombosis could be detected by IVUS before p53 gene transfection.The site of adenovirus injection was displayed by IVUS as the dent at the side of the vessel wall.3.Apoptosis One day after transfection,TUNEL staining showed apoptosis in 1.87%±0.17% of Ad5-CMV.lacZ-treated plaque cells as compared with 10.99%±0.78%of Ad5-CMV.p53-treated cells.However,14 days after transfection,the apoptosis rate was 0.97%±0.13%and 2.71%±0.42%in the Ad5-CMV.lacZ-treated group and Ad5-CMV.p53-treated group,respectively.Few apoptotic cells in the plaque of rabbits in group C were found which were located mainly in the lipid core (0.15%±0.07%).4.Adenoviral expression patternTo evaluate gene transfection efficiency and expression duration,β-galactosidase activity in Ad5-CMV.lacZ-treated plaques was measured and detected 1 day after transfection,both in the lesion core and fibrous cap,which was still detectable 14 days after transfection.In Ad5-CMV.p53-treated animals,p53-positive cells were localized mainly in the superficial,smooth muscle cell-rich layer of plaques.One day after Ad5-CMV.p53 transfection,a substantial number of p53-positive cells were identified in the intima.However,by day 14,p53 staining was minimal,p53 was not expressed at any time in Ad5-CMV.lacZ-treated or blank control plaques.5.Morphometry and immunohistochemical stainingAfter pharmacological triggering,of 13 Ad5-CMV.p53-treated rabbits,10 showed plaque rupture(76.9%)as compared with only 3 of 13 Ad5-CMV.lac Z-treated rabbits(23.1%,p＜0.01)or none in group C(0%,p＜0.01).In comparison with AdS-CMV.lacZ-treated plaques and blank control plaques,the decrease in the fibrous cap thickness in Ad5-CMV.p53-treated plaques was confirmed by decreased VSMC number and collagen-rich matrix on staining with Masson trichrome,α-actin staining and picrosirius red staining viewed under polarized light.This effect was seen to translate to a significant decrease in the cap/intima ratio in Ad5-CMV.p53-treated arteries.In the Ad5-CMV.p53-treated rabbits,there was a thinner fibrous cap,with focal accumulation of cap macrophages as confirmed by RAM11 immunostaining.While in both Ad5-CMV.lacZ-treated and blank control rabbits,plaques contained a thick VSMC-rich fibrous cap with abundant collagen, with definite macrophages scattered throughout the lesions.Bivariate correlation analysis showed good positive correlations between hs-CRP, MCP-1,sVCAM-1,sICAM-1,ox-LDL or apoptosis rate and plaque rupture(r=0.758, 0.681,0.663,0.579,0.590,0.603,respectively,all p＜0.01).In contrast,no correlation was found between TC,TG,LDL-c or HDL-C and plaque rupture(both p＞0.05).In order to compare the relative importance of apoptosis and inflammation in mediating plaque instability,a logistic regression model was applied in which the status of plaque rupture was the dependent variable and the apoptosis rate and inflammatory markers were entered as the independent covariables.The results showed that only serum level of hs-CRP had an independent effect on plaque rupture with odds ratios as 1.314(95%CI:1.041～1.657,p=0.021),suggesting that inflammation played an more important role in plaque instability than apoptosis.A linear regression analysis was performed to reveal the relationship between apoptosis rate and inflammatory markers.The results demonstrated good positive correlations between hs-CRP,MCP-1,sVCAM-1,sICAM-1,ox-LDL and apoptosis rate(r=0.761,0.557,0.616,0.734 and 0.691 respectively,all p＜0.01).ConclusionsWe have developed a new animal model of vulnerable plaques by direct injection of Ad5-CMV.p53 into atherosclerotic plaques,which offers advantages of targeted gene delivery,accurate dosing,high transfection efficiency,high plaque rupture rate and low mortality rate.The mechanisms of p53-mediated plaque instability may involve cell apoptosis in the fibrous cap,inflammation and oxidative stress.Cell apoptosis is the causative factor,inflammation-collagen metabolic regulation network disorder,involved inflammatory cells,inflammatory factors,smooth muscle cells and collagen synthesis and degradation,is the major molecular mechanism and thinner cap thickness is the major pathological mechanism of p53-induced plaque unstability. BackgroudThe rupture of atherosclerotic plaques and the following-up thrombogenesis is evidenced to be the main etiology of cardiovascular atherosclerotic disease.Early detection of vulnerable plaques plays a vital role on the triage of vulnerable patients and the active interference of cardiovascular disease.Moreover,the stability of plaques is a key basis of therapeutic effect evaluation.Inflammatory,immunologic, metabolic and thrombotic factors are all involved in the process from stable plaques to vulnerable plaques.Thus,challenges are raised for diagnostic techniques which image the mere silhouette of the artery lumen and atherosclerotic plaques.To evaluate the vulnerability,demonstrating the morphology and the functional states of plaques are essential.Vulnerability is the intrinsic factors for plaque rupture,inflammation-collagen metabolic regulation network disorder,involved inflammatory cells,inflammatory factors,smooth muscle cells and collagen synthesis and degradation,is the major molecular mechanism of plaque instability.Extrinsic factors,which can change the stress-strain state of the plaque,may favor the genesis of plaque rupture.The vulnerability of atherosclerotic plaques is determined by the size of lipid core,the thickness of fibrous cap and the extent of focus inflammation.Meanwhile,these factors are also the determinants of the molecular structures of plaques and the latter plays an important role on tissue elasticity.Both static and dynamic mathematic models have elucidated that drastic displacement or distortion of the plaque, especially the fibrous cap,is the key predictor of plaque rupture.Thus,the study of strain distribution patterns of atherosclerotic plaques has the potential value on screening vulnerable plaques,elucidating the mechanism of vulnerability and predicting the location of rupture.Strain,originally derived from Doppler tissue imaging,is a valuable parameter in the assessment of left ventricular regional contraction.However,Doppler technique is limited by its beam-to-motion angle dependency.Local strain of vessel wall can also be displayed as a color map(elastogram)superimposed on the intravascular ultrasonic images,but the invasive nature of this modality has prohibited its wide applications.Recently,velocity vector imaging(VVI),a tool for three-dimensional strain measurement from grayscale images based on multiple tracking techniques,has been introduced to determine tissue strain.Because the information derived by VVI is angle independent,VVI can be used to measure tissue motion perpendicular to the ultrasonic beam.Although VVI has been used successfully to measure myocardial strain,the role of this technique in tracking the motion and determining the strain of atherosclerotic plaques is uncertain.Objectives1.To test the hypothesis that the peak strain of atherosclerotic plaques can be reliably measured by VVI which may provide a useful index for detecting vulnerable plaques in a rabbit model ofatherosclerosis.2.To elucidate the biomechanical mechanism of plaque rupture based on the study of relationship between three-dimensional strain and inflammation-collagen metabolic regulation network of atherosclerosis plaque.Methods 1.Experimental protocolA rabbit model of vulnerable plaque was produced by the method in partâ… .A total of 60 male New Zealand White rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1% cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 3 groups: Ad5-CMV.p53 transfected group(n=20),Ad5-CMV.lac Z transfected group(n=20) and blank control group(n=20).At week 16 since the beginning of the experiment, under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53 (8×10~9pfu/ml)or lac Z(8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in the Ad5-CMV.p53 transfected group or Ad5-CMV.lac Z transfected group,respectively.Two weeks after transfection, plaque disruption was triggered by administration of Chinese Russell's viper venom (0.15 mg/kg,intraperitoneally),followed 30 min later by histamine(0.02 mg/kg intravenously)at 24 h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight at the end of week 18 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.3.Ultrasonographic studiesIVUS was accomplished at the end of week 16 to identify the most prominent aortic plaques and guide adventitial injection into the plaques.IVUS was repeated at the end of week 18 to measure the lumen area(LA),the external elastic membrane area(EEMA),plaque area(PA),the percentage of plaque burden(PB),eccentric index(EI)and remodeling index(RI).At the end of week 18 since the beginning of the experiment,the abdominal aortas were detected with high frequency duplex ultrasonographic system(HP SONOS 5500)and a 7.5-MHz transducer.The aortic longitudinal and transversal axis views were obtained;aortic diameter(D)and intima-media thickness(IMT)were measured.Doppler flow measurement was performed to derive the aortic peak velocity.Ultrasonic integrated backscatter(IBS)from the aortic wall and atherosclerotic plaques were analyzed by the acoustic densitometry technique.The average ultrasonic intensities(Aâ…¡)of aortic intima and aventitia in plaques of rabbits in all three groups,and the corrected Aâ…¡(Aâ…¡c%)was derived by calculating the ratio of Aâ…¡of the intima to Aâ…¡of the adventitia.At the same time,an ultrasound system(Acuson Sequoia C512)and a 15L8W transducer was carefully manipulated to image the largest plaque at the level of gene transfection.The long and short axis views of plaques were acquired with cine loops which were stored digitally on a magneto optic disk for offline analysis.The longitudinal peak strain(LSp),the circumferential peak strain(CSp)and the radial peak strain(RSp)of stable or unstable AS plaques in the rabbits of three groups were analyzed.4.Histological and immunohistochemical stainingSerial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),picrosirius red,oil red O,MOVAT and specific immunohistochemical staining.The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,andα-smooth muscle cell actin to detect VSMCs,collagen typeâ… ,collagen typeâ…¢to observe collagen distribution,MMP1,MMP2,MMP3,MMP9,TIMP1 and TIMP2 to view inflammatory response.The fibrous cap thickness,intima thickness and cap/intima ratio were measured and values averaged.The area of positive staining of lipids,collagen,VSMCs and macrophages was expressed as a percentage of staining area divided by plaque area in at least 10 high power fields(x400).The vulnerability index was calculated as (macrophage staining%+lipid staining%)/(SMCs%+collagen fiber%).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as fibrous cap discontinuity connected with overlying intraluminal thrombus. 5.Quantitative Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)The mRNA expression of various inflammation meditators MCP-1,ICAM-1, MMP-1,MMP-2,MMP-3,MMP-9,TIMP-1 and TIMP-2 in the abdominal arterial atherosclerosis lesions was determined.6.Statistical analysisAll numeric data were expressed as mean±SD and showed by one-sample Kolmogorov-Smimov test to be in normal distribution.Differences in continuous variables between two groups were assessed by unpaired t-test,and comparison among multiple groups was performed by analysis of variance with ANOVA. Categorical variables were analyzed by Fisher's exact test.All radial(RSp), circumferential(CSp)and longitudinal(LSp)peak strain values were multiplied by 100 and entered into a binary logistic regression model to screen risk factors predictive of plaque rupture.Receiver operating characteristic(ROC)analysis was performed to identify the optimal parameter and its sensitivity and specificity for detecting vulnerable plaques.Subsequently,linear regression analysis was performed to evaluate the relationship between peak strain and histological features including contents of macrophages,SMCs,collagens,lipids,vulnerability index and cap thickness.All data analysis was performed by SPSS version 13.0(SPSS Inc.,Chicago, USA).A p value＜0.05 was considered significant.Results1.Incidence of plaque ruptureHistological analysis indicated that after pharmacological triggering,of 20 Ad5-CMV.p53-treated rabbits,15 showed plaque rupture(75%)as compared with only 7 of 20 Ad5-CMV.lac Z-treated rabbits(35%,p＜0.01)and 3 of 20 blank control rabbits(15%,p＜0.01).In total,there were 25 rabbits with plaque rupture and 35 rabbits without plaque rupture after pharmacological triggering.2.Biochemical studies At the week of 18,no significant change of TC,TG LDL-c and HDL-C was found among the three groups(p＞0.05),however,the level of hs-CRP,MCP-1,sICAM-1,sVCAM-1 and ox-LDL in rupture group was higher than that in nonrupture group(p＜0.01).3.Ultrasound measurement:There were more eccentric plaques in rabbits with ruptured plaques than in those without,and the levels of EEMA,PA,PB in the ruptured group were also significantly larger than those without rupture(p＜0.01).Positive remodeling pattern was observed more frequently in rabbits with ruptured plaques,whereas normal and negative remodeling patterns were more common in rabbits without plaque rupture.IMT in rupture group was thicker than that in nonrupture group(p＜0.01).D in rupture group was significantly bigger than nonrupture group(p＜0.01).However, there was no significant change of Vp was found in both rupture group and nonrupture group(p＞0.05).Values of Aâ…¡c%in rupture group were significantly lower than that in nonruptured group.The RSp and CSp values were significantly higher in the Ad5-CMV.p53 transfected group than in the Ad5-CMV.lac Z transfected group or blank control group.In contrast,the LSp values were significantly lower in the Ad5-CMV.p53 transfected group than in the other two groups.Similarly,the values of RSp and CSp were higher whereas the values of LSp were lower in ruptured plaques than the corresponding values in non-ruptured plaques(p＜0.01).In order to test whether RSp,CSp and LSp can predict the occurrence of plaque rupture,we used a binary logistic regression model in which the status of plaque rupture was the dependent variable and the strain values were entered as independent predictors.The results showed that both RSp and CSp were significant predictors of plaque rupture with odds ratios of 1.285(95%CI:1.011～1.634,p=0.040)and 1.252 (95%CI:1.014～1.5479,p=0.037),suggesting that increased RSp and CSp were significant risk factors of plaque rupture.On the other hand,LSp was not a risk factor of plaque rupture with an odds ratio of 1.116(95%CI:0.991～1.256,p=0.069).ROC analysis revealed a sensitivity of 88.0%and 88.6%and a specificity of 88.6%and 92.0%with the area under the curve being 0.963 and 0.965 to predict plaque disruption at a cut-off value of 0.296%and 0.268%for RSp and CSp,respectively.4.Histological features and plaque strainThe fibrous cap thickness of plaques in the Ad5-CMV.p53 transfected group was significantly lower than in the Ad5-CMV.lac Z transfected group and blank control group.Oil red O staining demonstrated no significant difference with regard to lipid positive staining area among the three groups.Picrosirius red staining viewed under polarized light revealed that the collagen content of plaques was significantly lower in the Ad5-CMV.p53 transfected group than in the other two groups.Likewise,more positive SMCs actin staining was found in plaques of the Ad5-CMV.p53 transfected group than in the Ad5-CMV.lac Z transfected group or blank control group.As depicted by RAM11 immunohistochemistry,macrophage content was more extensive in plaques of the Ad5-CMV.p53 transfected group than the Ad5-CMV.lac Z transfected group or blank control group.Similarly,a statistically thinner fibrous cap, higher macrophage content and lower SMCs and collagen content were found in the ruptured plaques than in the non-ruptured plaques,which led to an increased vulnerability index in rabbits with disrupted plaques.The results of NF-κB,MMP-1, MMP-2,MMP-3,MMP-9 and collagen typeâ…¢immunostaining were similar to that of macrophage staining.The positive staining of collagen typeâ… ,TIMP1 and TIMP2 was lower in ruptured plaques than in nonruptured plaquesLinear regression analysis showed a good positive correlation between RSp or CSp and the macrophage content in plaques,and a negative correlation between RSp or CSp and the fibrous cap thickness or content of SMCs and collagen(all p＜0.01). As a result,there was a good correlation between RSp or CSp and plaque vulnerability index.In contrast,there was a negative correlation between LSp and the macrophage content in plaques,and a positive correlation between LSp and the fibrous cap thickness or content of SMCs and collagen(all p＜0.01).Consequently, there was a negative correlation between LSp and plaque vulnerability index.The pathologic results showed,inflammation-collagen metabolic regulation network,involved in inflammatory cells and MMPs induced collagen degradation enhancement and SMCs induced collagen synthesis reduction,was the major features of plaque vulnerability in this model.Statistic analysis showed that there were good correlations between RSp,CSp and macrophages,SMCs,collagen content of plaque, so RSp and CSp were a novel index of plaque vulnerability and also the pattern of manifestation of inflammation-collagen metabolic network.5.Quantitative Real-time RT-PCRThe relative mRNA expression of MCP-1,ICAM-1,MMP-1,MMP-2,MMP-3,MMP-9 was higher in rupture plaques than in nonrupture plaques(all P＜0.05).On the contrary,the TIMP-1 and TIMP-2 mRNA expression was lower in rupture plaques than in nonrupture plaques.Conclusions1.VVI is a new noninvasive technique that allows measurement of peak strain of plaques in a rabbit model of atherosclerosis.With the evolution of plaques from stable to unstable states,RSp and CSp increased while LSp decreased.RSp and CSp measured by VVI provided a novel index with a high sensitivity and specificity for detecting vulnerable plaques.2.Inflammation-collagen metabolic regulation network,involved inflammatory cells,inflammatory factors and collagen synthesis and degradation,leads to plaque cap thinner and lipid core larger,which increases local shear stress and makes circumferential stress transfer to the shoulders of plaques.Because local strain measures relative deformation in a given vascular segment,such a parameter reflects the net result of the local stress imposed on and the elastic property of the vessel wall. Plaque rupture occurs when external mechanical forces exceed the tensile strength of the fibrous cap.RSp and CSp were a novel index of plaque vulnerability and also the pattern of manifestation of inflammation-collagen metabolic regulation network. BackgroudRecent studies show that atherosclerosis is a chronic inflammatory disease that is caused by multiple processes,including infiltration of inflammatory cells, proliferation of smooth muscle cells,increase in extracellular matrix and thrombus formation.It has been well established that plaque rupture and subsequent intraluminai thrombosis is the most common cause of acute coronary syndromes.The key point of prevention of acute cardiovascular events is detection of vulnerable plaques and therapy of stabilizing plaques.Previous studies have demonstrated that distribution of vulnerable plaque is diffused,while the main feature of vulnerable plaque is focus inflammation,therefore, both local and systemic inflammation plays a vital role in the stability of plaques. Several investigators have also noted that the presence of more than one vulnerable plaque in patients with acute coronary syndrome,and therefore,general drug therapy should be the basis of treatment of vulnerable plaques.Unfortunately,however,an ideal drug for stabilizing vulnerable plaques is still lacking.Although statins have been demonstrated in animal studies to significantly decrease lipid contents and inflammatory cells and thicken the fibrous caps of plaques by LDL-c lowering and inflammation inhibiting effects.However,liver dysfunction as a side effect of statins caused some patients to withdraw from statin treatment.Consequently,drugs of traditional Chinese medicine Tongxinluo with low side effects and multiple targets are of high interest as alternatives.Objectives1.To test the hypothesis that the traditional Chinese medicine Tongxinluo has anti-atherosclerotic progression and plaque-stabilizing propertie in a rabbit model of vulnerable atherosclerosis plaques.2.To elucidate the molecular mechanism of the traditional Chinese medicine Tongxinluo in stabilizing vulnerable plaques.Methods1.Experimental protocolA rabbit model of vulnerable plaque was produced by the method in partâ… .A total of 75 male New Zealand White rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a high-cholesterol diet(1% cholesterol)for 10 weeks.Subsequently,the atherogenic diet was replaced by a regular diet for another 6 weeks.Then rabbits were randomly divided into 5 groups: group A was control group(without treatment n=15),group D1 was given Tongxinluo(0.15g/kg/d,n=15),group D2 was given Tongxinluo(0.3g/kg/d,n=15), group D3 was given Tongxinluo(0.6g/kg/d,n=15),and group E was given simvastatin(5mg/kg/d,n=15).At week 16 since the beginning of the experiment, under the guidance of IVUS,a 50-μl suspension of adenovirus containing p53 (8×10~9pfu/ml)was injected from the aortic adventitia into the largest plaque in the abdominal aorta in all groups,respectively.Two weeks after transfection,plaque disruption was triggered by administration of Chinese Russell's viper venom(0.15 mg/kg,intraperitoneally),followed 30 min later by histamine(0.02 mg/kg intravenously)at 24 h and 48h before euthanization.2.Biochemical studiesBlood was drawn from rabbits fasting overnight at the end of week 18 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MCP-1,sICAM-1,sVCAM-1 and ox-LDL.3.Ultrasonographic studiesAt the end of week 18 since the beginning of the experiment,the abdominal aortas were detected with high frequency duplex ultrasonographic system(HP SONOS 5500)and a 7.5-MHz transducer.The aortic longitudinal and transversal axis views were obtained;aortic diameter(D)and intima-media thickness(IMT)were measured.Doppler flow measurement was performed to derive the aortic peak velocity.Ultrasonic integrated backscatter(IBS)from the aortic wall and atherosclerotic plaques were analyzed by the acoustic densitometry technique.The average ultrasonic intensities(Aâ…¡)of aortic intima and aventitia in plaques of rabbits in all three groups,and the corrected Aâ…¡(Aâ…¡c%)was derived by calculating the ratio of Aâ…¡of the intima to Aâ…¡of the adventitia.At the same time,an ultrasound system(Acuson Sequoia C512)and a 15L8W transducer was carefully manipulated to image the largest plaque at the level of gene transfection.The long and short axis views of plaques were acquired with cine loops which were stored digitally on a magneto optic disk for offline analysis.The longitudinal peak strain(LSp),the circumferential peak strain(CSp)and the radial peak strain(RSp)of stable or unstable AS plaques in the rabbits of three groups were analyzed.IVUS was accomplished at the end of week 18 to measure the external elastic membrane area(EEMA),the lumen area(LA),plaque area(PA),the percentage of plaque burden(PB),eccentric index(EI)and remodeling index(RI).4.Histological and immunohistochemical staining Serial cross-sections underwent general histological staining with hematoxylin & eosin(H&E),picrosirius red,oil red O and specific immunohistochemical staining. The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,and a-smooth muscle cell actin to detect VSMCs,LOX-1, MMP1,MMP2,MMP3,MMP9,TIMP1 and TIMP2 to view inflammatory response.The fibrous cap thickness,intima thickness and cap/intima ratio were measured and values averaged.The area of positive staining of lipids,collagen,VSMCs and macrophages was expressed as a percentage of staining area divided by plaque area in at least 10 high power fields(x400).The vulnerability index was calculated as (macrophage staining%+lipid staining%)/(SMCs%+collagen fiber%).Sections stained with H&E were analyzed for disrupted plaques,histologically defined as fibrous cap discontinuity connected with overlying intraluminal thrombus.5.Quantitative Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)The mRNA expression of various inflammation meditators LOX-1,MCP-1, ICAM-1,MMP-1,MM-2,MM-3,MMP-9,TIMP1 and TIMP2 in the abdominal arterial atherosclerosis lesions was determined.6.Statistical analysisAll numeric data were expressed as mean±SD and showed by one-sample Kolmogorov-Smimov test to be in normal distribution.Diffe...
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