Evaluation Of Plaque Elastic Mechanics And The Effect Of Statin On Plaque Stability By Introvascular Ultrosound Elastography

BackgroundSince the last century, cardiovascular disease has become "number one killer" to human health. Atherosclerotic vulnerable plaque disruption with superimposed thrombosis is considered the main cause of acute cardiovascular events. The study of the characteristics of vulnerable plaque for early detection and treatment, is important in preventing acute cardiovascular events. Tissue elasticity as a kind of biomechanical parameters, is influenced predominantly by pathological and physiological process in human body. Studies have found that the pressure in the lipid core, arterial pressure and shear stress caused by blood flow act on the fibrous cap of atherosclerotic plaque together, which makes the plaque deformed and strain energy stored; When the differential pressure on both sides of the fibrous cap exceeds a certain critical value, the plaque will become instable and probably broken. From the perspective of biomechanics, under the same stress, the lower the pressure threshold withstanded by the plaque, the more vulnerable plaque was thought to be. Both the pressure threshold of statics model, and vulnerability frequency range of dynamic model, are largely depends on the properties of plaque elastic mechanics. It is said that the plaque with stable elastic mechanics, is not easy to burst, while the opposite means vulnerable plaques. The size of plaque elasticity can be reflected by the size of plaque deformation under the same stress. The deformation is called "strain" in the field of biomechanics. Therefore, measuring the strain of the plaque can reflect the elasticity of plaques, and then evaluate the stability of plaques. This is the basis of the evaluation of plaque mechanical stability by using shear strain and area strain in the study.Developed on the basis of intravascular ultrasound, intravascular ultrasound elastography (IVUSE) is a new technology for evaluating the properties of plaque elastic mechanics. IVUSE is built according to the two kinds of theories:one kind is based on radio frequency signal, the other is based on digital image. The latter IVUSE through analyzing the images before and after its deformation, can quickly calculate the tissue strain and construct the elastic graph. Recent studies showed that IVUSE could be applied in the identification of vulnerable plaques.From a biomechanical point of view, vulnerable and stable atherosclerotic plaques are distinguished by heterogeneous living materials with peculiar mechanical properties depending on geometry, composition, loading and boundary conditions. Our previous research by using an in-house-designed IVUSE software system has confirmed that plaque tissue components has relationship with its mechanical properties, and also proved that the software is reliable in assessing the plaque elastic mechanics features. At present, some studies have observed plaque geometry is associated with the stability of the plaques:eccentric plaque is more vulnerable than centripetal plaque; the plaque causing vessel obviously narrow is more likely to rupture; vessel with positive remodeling is in the higher incidence of plaque rupture. However, there are also some studies observed contradictions, e.g. the criminal plaques are not usually at the most narrow vascular site. These findings from prospective observational studies are inconsistent, and the mechanisms are lack of adequate explanation. This study was aimed to research the elastic mechanical properties of atherosclerotic plaques with different morphological properties by using IVUSE, and explain the morphological features of vulnerable plaques from a biomechanical point of view.Objectives1. To investigate the elastic mechanical properties of atherosclerotic plaques with different morphological properties.2. To explain the morphological features of vulnerable plaques from a biomechanical point of viewMethods1. Animal preparation30 purebred male New Zealand rabbits after 2 weeks of atherogenic diet (1% cholesterol), underwent balloon-induced abdominal aortic endothelium injury, to accelerate the formation of atherosclerotic plaques. Postoperatively,400 thousand units of penicillin intramuscular injected to prevent infection. All the animals received the atherogenic diet until the end of week 12.2. IVUS examination and IVUSE acquisitionAt the end of week 12, abdominal aortas were first scanned by use of the IVUS system with the assistance of an automatic pullback device at 0.5 mm/s, then 2 plaques with moderate echo were chosen in at least 1-cm intervals from each rabbit for in situ imaging for at least 3 cardiac cycles.IVUSE involved an in-house-designed software system. Two consecutive frames near end-diastole from the IVUS cine in situ recognized as the pre-and post-deformed image, respectively, were used to generate the IVUSE.3. Measurement of Conventional and IVUSE VariablesThe IVUS pullback images and cross-sectional views in situ were used to determine the contours of atherosclerotic plaques.Conventional variables include:external elastic membrane area (EEMarea), lumen area (Lumenarea), Cross-sectional plaque area (PA), Plaque burden (PB), external elastic membrane volume (EEMvolume), lumen volume (Lumenvolume), plaque volume (PV), plaque volume burden (PVB), minimal plaque thickness (PTmin), maximal plaque thickness (PTmax), eccentric index (El), remodelling index (RI).IVUSE variables include:Shear strain (SS) and area strain (AS).4. Grouping1) Eccentric plaque group:E1> 0.5, Centripetal plaque group:El< 0.5;2) Eccentric plaque I group:E1> 0.75, Eccentric plaque Ⅱ group:0.5< E1 ≤0.75;3) Low plaque burden group:PB≤40%, High plaque burden group:PB> 40%;4) Positive remodeling group:RI> 1.05, Negative remodeling group:RI< 0.95, No remodeling group:0.95< RI< 1.05.5. Statistical AnalysisStatistical analysis involved use of IBM SPSS 19.0. All quantitative variables are expressed as mean ± SD for normally distributed variables and median (interquartile range) for nonparametric data. The Shapiro-Wilk test was used to assess the normality of quantitative variables. The homogeneity of variances was examined with the Levene test. Between-group comparisons involved the Student t test, Welch’s t test or Mann-Whitney U test as appropriate. One-way ANOVA and Kruskal-Wallis were used to compare the differences of multiple groups as appropriate. Correlations of shear and area strain with morphologic data were evaluated by stepwise multiple linear regression analysis. Two-tailed p< 0.05 was considered statistically significant (When nonparametric test was used in comparison between any two means of multiple groups, two-tailed p< 0.017 was considered statistically).Results1. Five rabbits died during the experiment, and data for two plaques were eliminated because of poor ultrasound images. Finally, data for 48 plaques were analyzed.2. The differences of elastic mechanics between eccentric plaques and centripetal plaquesIn contrast, the eccentric plaques showed obviously less PA, PB, PV and Tmin (p<0.05), but significantly greater El, SS and AS than the centripetal plaques (p<0.05).3. The elasticity characteristics of eccentric plaques with EI≤75%In eccentric plaque I group, the PA, PB, PV, PVB, Tmin and Tmax were significantly less than in eccentric plaque II group, but El and SS greater (p<0.05). In eccentric plaque I group, the PA, PB, PV, PVB and Tmin were also less than in centripetal plaque, but El, SS and AS apparently greater (p<0.05); All the parameters except for Tmin and El (p<0.05). The AS value of eccentric plaque II was in the middle level between the eccentric plaque I and concentric plaque, but no significant differences were found.4. The relationship of plaque burden and elastic mechanical propertiesThe low burden plaques showed less PA, PV, PB, PVB, Tmin and Tmax, and greater Lumenarea, Lumenvoiume and El than the high burden plaques (p<0.05). There is no difference in SS and AS between groups. In eccentric plaques I group, the low burden plaques had greater Lumenarea and Lumenvolume than the high burden plaques, but had less PA, PB, PVB, Tmax, and SS and AS than the high burden plaques (p<0.05).5. The relationship of vascular remodeling and plaque elastic mechanical characteristicsThe remodeling index was significant different in three groups (p<0.05). The SS and AS of plaques were significantly greater in negative remodeling group than in no remodeling group (p<0.05).6. The influence factors of plaque elastic mechanical propertiesStepwise multiple linear regression analysis showed that the plaque SS and AS had relationship with PB and El, and the impact of El on SS and AS was larger. Regression equations are:y=-5.119+6.330x1+8.929x2 (y:SS; x1:E1; x2:PB), R2=0.365, F=12.928, df= 47, p=0.000 and y=-7.677+10.050x1+14.157x2 (y:AS; x1: El; X2:PB), R2=0.359, F= 12.616, df= 47, p=0.000, respectively. There were significant parameter estimates in regression models (p<0.01)Conclusions1. The study found the relationship between plaque morphology and strain:plaques associated with eccentricity, high burden and negative remodeling perform greater strain than with centripetalism, low burden and no remodeling.2. Plaque eccentric index and plaque burden can be used to predict the elastic stability of plaques, and eccentric index has greater influence on elastic stability of plaques.Backgroud3-hydroxy-3-methylglutaryl Co A reductase inhibitors (statins) are among the first-line pharmacotherapeutic agents for atherosclerosis treatment. Statins reduce the progression of atherosclerosis or even decrease the volume of atherosclerotic plaques. Plaque volume or the consequent severity of stenosis produced might be a predictor of acute cardiovascular events, but the occurrence of events is affected simultaneously by many elements. Plaque vulnerability is considered the most important determinant of acute cardiovascular events.Thrombosis secondary to rupture of vulnerable plaque is the main cause of acute cardiovascular events. Plaque of recognition and treatment is important for prevention of acute cardiovascular event. Characteristics of vulnerable plaques are large lipid pools, thin fibrous cap, less inflammatory cells, smooth muscle cells. Therefore, the morphological features alone cannot meet the needs for evaluation of rosuvastatin plaque. A precise functional measure that can represent a diagnostic technology for plaque vulnerability in vivo is needed. Intravascular ultrasond elastography (IVUSE) provide an accurate evaluation of elasticity characteristica of plaques, and many studies have used this technique for identification of vulnerable plaque. Our previous studies showed a good association of plaque deformation and its tissue features by means of an in-house-designed FVUSE software system. However, IVUSE has not been used for assessing the effect of statins on the mechanical properties of atherosclerotic plaques. Here, we used IVUSE to investigate the mechanical properties of atherosclerotic plaques in a rabbit model with rosuvastatin treatment.Objectives1. To evaluate the effect of rosuvastatin on the elastic mechanical properties of rabbit atherosclerotic plaque by IVUSE.2. To expound the mechanism which lie in the effect of rosuvastatin on mechanical properties of atherosclerotic plaques.Methods1. Animal Preparation20 purebred male New Zealand rabbits underwent abdominal aortic endothelium denudation with an intravascular balloon catheter (4.0 x 15 mm) after 2 weeks of an atherogenic diet (1% cholesterol). At week 13, rabbits were randomly divided into 2 groups (n=10 each) for treatment:rosuvastatin (1.5 mg/kg/day) or equal volume of saline (control) for 8 weeks. At the end of week 20, animals were killed and abdominal aortas were harvested.2. Plasma AssaysBefore treatment and at sacrifice, a blood sample in fasting state was drawn from the rabbit ear margin vein. After 8-min centrifugation at 4000 rpm, plasma levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured.3. IVUS Examination and IVUSE AcquisitionBefore and after rosuvastatin or saline administration, abdominal aortas were first scanned by use of the IVUS system with the assistance of an automatic pullback device at 0.5 mm/s, then 2 plaques were chosen in at least 1-cm intervals from each rabbit for in situ imaging for at least 3 cardiac cycles. The distance of plaques from the end of the abdominal aorta were recorded.IVUSE involved an in-house-designed software system. Two consecutive frames near end-diastole from the IVUS cine in situ recognized as the pre-and post-deformed image, respectively, were used to generate the IVUSE.4. Measurement of Conventional and IVUSE VariablesThe IVUS pullback images and cross-sectional views in situ were used to determine the contours of atherosclerotic plaques.Conventional variables include:external elastic membrane area (EEMarea), lumen area (Lumenarea), Cross-sectional plaque area (PA), Plaque burden (PB), external elastic membrane volume (EEMvolume), lumen volume (Lumenvolume), plaque volume (PV), plaque volume burden (PVB), minimal plaque thickness (PTmin), maximal plaque thickness (PTmax), eccentric index (EI), remodelling index (RI).IVUSE variables include:Shear strain (SS) and area strain (AS).5. Histopathology and ImmunohistochemistryAfter the perfusion with 10% Ringer formaldehyde solution, abdominal aortas were segmented according to the distance from the end of the abdominal aorta recorded during the IVUS detection. All aorta segments were soaked in 10% Ringer formaldehyde solution for 48 hr, then each segment was divided into two parts. One part was frozen, embedded and cut into serial slices of 6 μm for Oil-red O staining, and the other part was embedded in paraffin and cut into serial slices of 4 μm for picrosirius red staining and immunohistochemistry.6. TUNEL StainingThe aorta segments were soaked in 10% Ringer formaldehyde solution for 48 hr, then embedded in paraffin and cut into serial slices of 4 μm. The number of TUNEL-positive cells in the plaques was indicated by using the Plus Fluorescein In Situ Apoptosis Detection Kit (ApopTag, EMD Millipore Corp., USA), according to the manufacturer’s instructions. Apoptosis ratio was calculated as TUNEL-positive cells number divided by total cells number in 5 high-power fields (x400).7. Statistical AnalysisStatistical analysis involved use of IBM SPSS 19.0. All quantitative variables are expressed as mean ± SD for normally distributed variables and median (interquartile range) for nonparametric data. The Shapiro-Wilk test was used to assess the normality of quantitative variables. The homogeneity of variances was examined with the Levene test. Differences between paired continuous data were assessed by paired Student t test or Wilcoxon test as appropriate. Between-group comparisons involved the Student t test, Welch’s t test or Mann-Whitney U test as appropriate. Correlations of shear and area strain with VI and plaque components were evaluated by Pearson’s correlation coefficient. Two-tailed p< 0.05 was considered statistically significant.Results1. General Rabbit Characteristics and Plaque Morphological PropertiesFive rabbits died during the experiment, and data for 1 rabbit were eliminated because of poor ultrasound images. Finally, data for 14 rabbits were analyzed; each group involved 7 rabbits.Rosuvastatin therapy decreased serum TC and LDL-C as well as the plaque EEMarea, Lumenarea, EEMvolume and Lumenvolume (p<0.05), with unchanged serum TG and HDL-C levels as well as plaque PA, PB, PV, PVB, PTmax and E1. In comparison, serum TC and LDL-C levels as well as plaque PA, PB, PV, PVB and EEMvolume increased with time in the control group (p<0.05). Differences in changes in serum TC and LDL-C as well as the plaque EEMarea, Lumenarea, EEMvolume, Lumenvolume, PA, PV and PVB between the rosuvastatin and control groups on serial follow-up were significant (p<0.05). The rosuvastatin group showed negative remodeling and the control group slightly positive remodeling, with a significant difference between groups (p<0.001).2. Plaque Mechanical PropertiesRosuvastatin rabbits showed a stable magnitude of SS and AS by IVUSE in all 14 plaques but also in the total, shoulder and body regions of the 11 eccentric plaques. In contrast, control rabbits showed a significant increase in SS and AS in all 14 plaques (p<0.01). Similar changes were observed in 9 eccentric plaques of control rabbits and also in shoulders of 9 eccentric plaques (p<0.05) but not in plaque body. On serial follow-up, rosuvastatin and control groups differed in changes in SS and AS for total plaques (p<0.05). As well, the changes in SS and AS significantly differed between groups for total eccentric plaques and in shoulders (p<0.05). At follow-up, in the control group, eccentric plaques showed greater SS and AS in the shoulder than body (p<0.05).3. Plaque Tissue ComponentsRelative areas of collagen in plaques were larger with rosuvastatin than control treatment (p<0.01), whereas relative areas of macrophages and lipids were smaller with rosuvastatin than control treatment (p<0.05). Rosuvastatin substantially reduced the plaque VI as compared with control treatment (p<0.01).4. Differences in Proinflammatory Cytokines Levels and Apoptosis between Groups Control plaques showed large levels of MCP-1, MMP-2 and TNF-a., which were decreased significantly in rosuvastatin-treated plaques (p<0.05). Immunohistochemical stain revealed increased content of MMP-2 at the plaque thickened intimal layer, especially under the fibrous cap in the shoulder of the plaque. Rosuvastatin treatment reduced apoptosis in atherosclerotic plaques as compared with control treatment (p<0.05).5. Correlations Between SS and AS and Plaque Components or VISS showed moderately negative correlations with collagen content (p=0.001) and SMC content (p<0.01) and moderately positive correlations with macrophage infiltration (p<0.01) and lipid content (p< 0.001) as well as VI (p< 0.001). AS showed similar correlations with collagen content (p<0.01), SMC content (p<0.01), macrophage infiltration (p<0.05) and lipid content (p= 0.001) as well as VI (p< 0.001). SS showed moderately positive correlations with TNF-a level (p< 0.05) and apoptosis ratio (p< 0.05) as did AS with TNF-a level (p< 0.05) and apoptosis ratio (p < 0.05).Conclusions1. Our findings revealed that treatment with rosuvastatin in a rabbit model stabilized atherosclerotic plaques by changing its histic and molecular composition.2. Rosuvastatin can effectively control the shear strain and area strain in the shoulder of eccentric plaque which showed more unstable mechanical properties.3. The differences in mechanical properties were related to plaque composition, inflammatory reaction and apoptosis.4. IVUSE is a very sensitive technique for detecting pharmacologically-induced mechanical changes in rabbit atherosclerotic plaques.