Establishment Of A Vulnerable Plaque Animal Model And Gene Therapy For Stabilizing Vulnerable Plaque

BackgroundAtherosclerotic plaque rupture has been identified as the major cause of acute coronary syndromes (ACS). Vulnerability is the intrinsic factors for plaque rupture. A plaque with a lipid pool and a thin, weakened fibrous cap infiltrated by macrophages is defined as vulnerable plaque. Extrinsic factors, which can change the stress-strain state of the plaque, may favor the genesis of plaque rupture.It is very important to recognize and prevention the acute cardiovascular events as early as possible. 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 mechanism of vulnerable plaque as well as the studies of prevention and treatment would be greatly aided by the availability of an animal model. However, it is still not satisfying with the present animal model. There are several limitations. Firstly, atherosclerotic process in the animal models is not histologically identical to that in humans. Secondly, inflammation in the plaque is not intense. Thirdly, spontaneous plaque rupture rate is low and ruptured plaque accompanied by the formation of platelet-rich fibrin thrombi was rare.In addition, most of the previous studies typically focus on histological in vitro examinations. Recently, noninvasive imaging techniques were used for detect the development of atherosclerosis plaque in apolipoprotein E knockout (apoE-/-) mice. Whether it can be used for identifying vulnerable plaque in the early stage is limited.In the present study, we aimed to establish an animal model of spontaneous plaque rupture in apoE-/- mice under a high-fat diet by a combination of mental stress and lipopolysaccharide (LPS) treatment, and elucidate the mechanisms of plaque vulnerability in such kind of animal model. At the same time, micro-ultrasound imaging would be conducted for examining the morphology and hemodynamics of carotid vulnerable plaques of apoE-KO mice, and the feasibility of the technique in detecting plaque vulnerability in mice in vivo would be evaluated. Objectives1. To establish an animal model of spontaneous plaque rupture in high-fat diet apoE knockout mice by combination of mental stress and Lipopolysaccharide (LPS) treatment. Plaque morphology and compositions and the interactive effects of stress and LPS on plaque vulnerability were analyzed.2. To elucidate the pathophysiological and molecular mechanisms of multiple-factor-induced plaque rupture in apoE-/- mice.3. To evaluate the values of micro-ultrasound imaging techonology on detection of vulnerable plaque in apoE-/- mice.Methods1. Animal ProtocolMale apoE-/- mice (n=108), 10 to 12 weeks of age, were randomly divided into four groups, including Stress group, LPS group, Stress/LPS group and control group (27 for each group). Carotid atherosclerotic lesions were induced by perivascular constrictive collars placement on the left common carotid arteries.¹³ Four weeks after surgery, mice in LPS group and Stress/LPS group were injected intraperitoneally with LPS (lmg/kg, Sigma, USA) twice per week for eight weeks. Eight weeks after surgery, mice in Stress group and Stress/LPS group were administered a one-hour electric foot-shock (0.5 mA for 5-second period every 3 minutes) and noise (HOdB for 3-second period every 5 minutes) stress by an stimulator every day for four weeks. Mice in control were not administrated any stimulation. 2. Hemodynamic AssessmentAt the end of the experiment, systolic (SBP) and diastolic blood pressure (DBP) and heart rates (HR) was measured. Micro-ultrasound Imaging Measurement was performed to measure the maximal velocity (Vmax) of carotid blood flow and left ventricular ejection fraction (LVEF) for assessing hemodynamic status of the body.3. Micro-ultrasound Measurement Imaging characteristics of ruptured and nonruptured plaques were compared with micro-ultrasound imaging techonology. Plaque area, plaque length, external elastic membrane area (EEMA), the maximal and minimal intima-media thickness (IMT) and flow velocities were measured. Eccentric index (EI) and remodeling index (RI) were calculated.4. Blood Norepinephrine and Biological MeasurementsImmediately after the last stress stimulation to mice, blood samples were taken from retro-orbital bleeding. Serum norepinephrine (NE), high sensitive C-reactive protein (hsCRP), lipid and plasma fibrinogen was measured.5. Histological and Morphology AnalysesSections were stained with hematoxylin and eosin (H&E), Masson's trichrome, Sirius red staining, Verhoeff staining, Oil red O staining, Perl's staining. Smooth muscle cells (SMCs) and macrophages were detected by immunostaining with anti-a-actin and MOMA-2 antibody. Plaque area, vessel area and cap thickness were measured Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimal areas were calculated. Plaque rupture rate and vulnerable index were accounted.6. Real-Time RT-PCRThe mRNA levels of cytokines (including IL-6, IL-18, TNF-α, MCP-1) and matrix metalloproteinases (MMPs) (including MMP2, MMP9) were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology. Results1. Body WeighIn total, only one mouse in Stress/LPS group died after the first LPS injection. There were no significant differences in the body weights between groups (Table 2).2. Hernodynamic State AssessmentFactorial ANOVA revealed significant main effects for stress on SBP, DBP, HR, Vmax and LVEF (all P<0.001). SBP, DBP, HR, Vmax and LVEF were significantly higher in Stress and Stress/LPS groups than in LPS (all P < 0.001) and control groups (all P< 0.001).3. Micro-ultrasound MeasurementMicro-ultrasound imaging and corresponding cross-sectional histopathology data revealed positive correlations for plaque area(r=0.606, p=0.004), intima-medial thickness (IMT)(r=0.777, p<0.001), eccentric index (EI)(r=0.784, p<0.001) and remodeling index (RI)(r=0.854, p<0.001). Ultrasound-derived EI and RI in the interventional group were significantly greater than those in the control group (p=0.046 and p=0.006, respectively). Similarly, Ultrasound-derived IMT, EI and RI in the ruptured plaques were significantly greater than those in the nonruptured plaques (p<0.001,p<0.001 and p=0.002, respectively). Maximal flow velocity (Vmax) was higher in the ruptured plaque sites compared with nonruptured plaques sites (p<0.001). Multivariate logistic regression analysis revealed that IMT and Vmax were independent predictors of plaque rupture with their odds ratio as 1.017 and 1.005, respectively.4. Biological Parameters MeasurementThere was a significant main effect of stress on NE (P<0.001, Factorial ANOVA), but not for LPS. Serum NE levels in Stress and Stress/LPS group both significantly greater than that in LPS group (both P<0.001) and control (both P<0.001). Both stress and LPS significantly elevated serum hsCRP level (both P<0.001, Factorial ANOVA). Combination of stress and LPS treatment resulted in a markedly increase in serum hsCRP compared with the stress (P<0.001) and LPS (P<0.001) treatments alone. There were no significant differences in the lipid profile between groups. Both stress and LPS significantly increased plasma fibrinogen level (both P<0.001, Factorial ANOVA). Plasma fibrinogen from Stress/LPS group was higher than Stress group (P<0.001) and LPS group (P<0.001).5. Plaque MorphologyRupture rates from Stress and Stress/LPS group were significantly higher than control (P=0.036, 0.036, respective). Among the 7 cases of ruptured plaques in Stress/LPS group, 2 cases showed extensive luminal thrombus secondary to cap disrupture and one case showed intraplaque hemorrhages. No differences in the percentage of plaque area between groups were found. Both stress and LPS markedly decreased cap thickness (both P<0.001, Factorial ANOVA), furthermore, combination of stress and LPS resulted in a synergistic decrease in the cap thickness (P=0.048, Factorial ANOVA). Mean cap thickness from Stress/LPS group was statistically less than other three groups (all P<0.01). Likewise, both stress and LPS decreased cap/core ratio (both P<0.001, Factorial ANOVA), and combination of stress and LPS synergistic decreased cap/core ratio (P=0.019, Factorial ANOVA). Plaques in Stress/LPS group showed significant lower collagen content (P<0.01 for all), higher lipid (P<0.01 for all) and macrophage (P<0.05 for all) compared with other three groups. Factorial ANOVA analysis revealed significant interaction between stress and LPS on macrophage and collagen contents (P=0.013, 0.043, Factorial ANOVA). No differences of α-SMCs could be detected among groups (P>0.05). Vulnerable index from Stress/LPS group was 2.53±0.54, significantly greater than other three group (control: 0.85±0.07, P<0.001; LPS: 1.81±0.14, P=0.001; Stress: 1.78±0.12, P=0.001).6. Effects of Stress and LPS on Gene Expression within the LesionsFactorial ANOVA showed that stress significant increased IL-6 (P=0.001), IL-18 (P=0.01), TNF-α (P=0.044), ICAM-1(P=0.018),MCP-1 (P<0.001), TF (P=0.014), MMP9 (P=0.007) and MMP12 (P=0.039) expression, and LPS significant increased IL-6 (P=0.030), ICAM-1 (P=0.022),TF (P=0.049) and MCP-1 (P=0.017) expression. No interactions of stress and LPS on genes expression were found.Cytokines and MMPs expression in Stress/LPS plaques were all at a higher level compared with control (all P<0.05,). In addition, MCP-1 and MMP9 expression in Stress/LPS plaques were both higher than in LPS plaques (P=0.004, 0.018, respectively). Conclusions1. In the present study, we established an animal model with vulnerable plaque phenotype. The animal model was generated by perivascular constrictive collar placement around the carotid artery of high-fat diet apoE-/- mouse, followed by mental stress and LPS co-treatment. High frequency of spontaneous plaque rupture and thrombus, intraplaque hemorrhage are common in this kind of model.2. The combination of stress and LPS contributes to a high frequency of spontaneous plaque rupture with a high prevalence of thrombosis on carotid lesions of apoE-/- mice by heightening hemodynamic reactivity, blood clotting, and inflammation processes. Stress caused hemodynamic status changed reflected by increased SBP, DBP, HR, LVEF and Vmax. In the meanwhile, stress and LPS elevated plasma fibrinogen and formed a hypercoagulable state. Combination of stress and LPS contributed to intense inflammation showed by higher level of serum hsCRP and more inflammatory cytokines and MMPs expression in the plaques, which is vital to plaque vulnerability.3. micro-ultrasound imaging provides a reliable approach to the noninvasive and quantitative assessment of carotid plaques in apoE-KO mice, and IMT and Vmax were independent predictors of plaque rupture in this animal model. BackgroundAtherosclerosis is a chronic inflammatory disease that is caused by multiple processes, including infiltration of inflammatory cells, proliferation of smooth muscle cells, extra- cellular matrix accumulation and thrombus formation. Monocytes/macrophages is observed in the early stage of atherosclerotic lesions. The inflammatory cells locating in the plaque contribute to severe inflammation and increase plaque vulnerability by synthesizing and secreting a great number of cytokines. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine for recruitment and activation of monocytes/macrophages. The most prominent function of MCP-1 is to recruiting blood monocytes to the damaged endothelium, which is crucial for inflammation and plaque unstable. Blockade of the MCP-1/CCR2 pathway markedly limited progression and destabilization of established atherosclerotic lesions.The techonology of gene silence, which can be called RNA interference (RNAi), is an alternative experimental method of specific inhibition of target gene expression, and has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21-23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability.The studies of the effects of RNAi on atherosclerotic vulnerable plaque in vivo were rare so far. In the present study, we aimed to test whether MCP-1 gene expression can be effectively inhibited by shRNA transfer in vivo and the effects of MCP-1 gene silence on plaque characteristics should be analyzed. Furthermore, we would elucidate the mechanism of MCP-1 gene silence for stabilizing vulnerable plaque. Objectives1. To construct shRNA targeting mouse MCP-1 gene with RNAi techonology, and construct pGSadeno-shRNA with adenovirus as a vector.2. pGSadeno-shRNA was transferred into vulnerable atherosclerotic plaques of apoE-/- mice. Plaque characteristics were analyzed.3. Elucidate the effects of MCP-1 gene silence on vulnerable plaque and the corresponding mechanisms.Methods1. Establishment of pGSadeno-shRNADesigned and synthesized shRNA targeting mouse MCP-1 gene by RNAi techonology. Adenovirus-mediated pGSadeno-shRNA was conducted, which binded a green fluorescence protein. Vector of pGSadeno-HK was conducted as control.2. Animal modelAnimal model with AS vulnerable plaque was established by combination mental stress and LPS stimulation. Eighty male apoE-/- mice, 10 to 12 weeks of age, were received a Western-type diet (0.25% cholesterol and 15% cocoa butter) from the beginning of the study. Carotid atherosclerotic lesions were induced by perivascular constrictive collars placement on the left common carotid arteries. Four weeks after surgery, mice were injected intraperitoneally with LPS (1mg/kg) twice per week for eight weeks. Eight weeks after surgery, mice were administered a one-hour electric foot-shock (0.5 mA for 5-second period every 3 minutes) and noise (110dB for 3-second period every 5 minutes) stress by an stimulator every day for four weeks.3. pGSadeno-MCPl-shRNAtransfection in vivoTen weeks after perivascular collar placement surgery, mice were randomly divided into RNAi group (n=40) and control (n=40). Mice in RNAi group were transfected with pGSadeno-shRNA in the site of carotid plaque, while mice in control were transfected with pGSadeno-HK. Three days later, 1 mouse of RNAi group was sacrificed and the expression of green fluresence protein was observed. All the mice were sacrificed to detect plaque morphology and MCP-1 protein expression fourteen days later after the transfection.4. Blood lipid profileBlood samples were taken from retro-orbital bleeding. Serum lipids were measured.5. Histological and Morphology AnalysesSections were stained with hematoxylin and eosin (H&E), Masson's trichrome, sirius red staining, Verhoeff staining, oil red O staining, Perl's staining. Immunostaining were performed for detecting SMCs, macrophages, MCP-1, MMP-2, MMP-9 and MMP-12 expression. Plaque area, vessel area and cap thickness were measured. Smooth muscle cell, macrophage, lipid and collagen positive areas were quantified and the ratios correlated to the intimal areas were calculated. Plaque rupture rate and vulnerable index were calculated. 6. Real-Time RT-PCRThe mRNA levels of MCP-1 in the fresh carotid lesions.were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.7. Western-blotWestern-blot was performed for examining MCP-1 protein expression in the fresh carotid lesions. Results1. Establishment of pGSadeno-shRNAPlasmid of pGenesil-1-shRNA-MCPl, which binded the the sequence of EGFP and mouse MCP-1 gene, was conducted by RNAi techonology and transfected into HK293 cells. Forty-eight hours later, MCP-1 gene expression decreased 83.3% in cells transfected with shRNA-MCPl. Adnovirus-mediated pGSadeno-MCPl-shRNA was successfully constructed confirmed by PCR reaction.2. pGSadeno-shRNA transfection in vivoThree days after transfection, expression of green fluresence protein was 39.26% in RNAi plaque, and decreased to 20.28% fourteen days after transfection.3. Blood lipid profileNo statistic differences of serum lipid profiles were found brtween RNAi group and control.4. Histological and Morphology AnalysesAtherosclerotic plaques of fifteen animals of each group were analyzed for histology. No statistic difference of plaque area was found brtween RNAi group and control.(P-0.582). Compared with control, mean fibrous cap thickness (8.48±2.62 vs 6.44±1.07um; P=0.035), cap/core ratio (0.09±0.03 vs 0.06±0.02; P=0.042) and collagen (18.67%±4.43% vs 10.11%±4.08%; P=0.003) were significantly increased, and lipid content was significantly decreased (13.16%±1.66% vs 19.04%±3.07%; P=0.0015) in RNAi group. Plaque rupture rates were 13.33%, significantly lower than that in RNAi group (60%, P=0.01). No thrombus and intraplaque hemorrhage were observed in RNAi group. Immunostainning results showed that there were more α-SMC-actin (10.30%±2.92% vs 6.75%±1.16%; P=0.011) and less macrophages in the RNAi group (8.19%±3.68% vs 14.05%±2.61%; P=0.006) than those in control. MCP-1 protein expression reduced 69.7% in RNAi group compared with that in control (4.44%±0.73% vs 14.65%±3.46%; P<0.001). In addition, MMP-2, MMP-9 and MMP-12 protein expression were all significantly decreased in RNAi group than those in control (all P<0.05). Vulnerable index of RNAi group was statistically lower than that of control (0.78±0.32 vs 2.19±0.94, P=0.006).5. Real time RT-PCRThe mRNA expression of MCP-1 in the RNAi carotid plaques decreased 76.5% compared with that in controls (P=0.03).6. Western-blot analysisThe proteins expression of MCP-1 in the RNAi carotid plaques decreased 66.7% compared with that in controls (0.24±0.01 vs 0.72±0.03, P<0.001). Conclusions1. Adnovirus-mediated pGSadeno-shRNA was successfully conducted by RNAi techonology.2. Experiment of pGSadeno-shRNA transfection in vivo in apoE-/- mice showed that MCP-1 gene silence may stabilize vulnerable plaque by inhibiting inflammation of the plaque demonstrated by decreased macrophages, MMPs and NFkB expression.